Biodegradation of 4-chlorophenol by acclimated and unacclimated activated sludge—Evaluation of biokinetic coefficients

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Environmental Research 99 (2005) 243–252 www.elsevier.com/locate/envres

Biodegradation of 4-chlorophenol by acclimated and unacclimated activated sludge—Evaluation of biokinetic coefficients Erkan Sahinkaya, Filiz B. Dilek Department of Environmental Engineering, Middle East Technical University, Inonu Bulvari, 06531 Ankara, Turkey Received 2 July 2004; received in revised form 2 November 2004; accepted 11 November 2004 Available online 4 January 2005

Abstract Unacclimated and acclimated activated sludges were examined for their ability to degrade 4-CP (4-chlorophenol) in the presence and absence of a readily growing substrate using aerobic batch reactors. The effects of 4-CP on the m (specific growth rate), COD removal efficiency, Y (yield coefficient), and q (specific substrate utilization rate) were investigated. It was observed that the toxicity of 4-CP on the culture decreased remarkably after acclimation. For example, the IC50 value on the basis of m was found to increase from 130 to 218 mg/L with the acclimation of the culture. Although an increase in 4-CP concentration up to 300 mg/L has no adverse effect on the COD removal efficiency of the acclimated culture, a considerable decrease was observed in the case of an unacclimated culture. Although 4-CP removal was not observed with an unacclimated culture, almost complete removal was achieved with the acclimated culture, up to 300 mg/L. The Haldane kinetic model adequately predicted the biodegradation of 4-CP and the kinetic constants obtained were qm ¼ 41:17 mg=ðg MLVSS hÞ; K s ¼ 1:104 mg=L; and K i ¼ 194:4 mg=L: The degradation of 4-CP led to formation of 5-chloro-2-hydroxymuconic semialdehyde, which was further metabolized, indicating complete degradation of 4-CP via a meta-cleavage pathway. r 2004 Elsevier Inc. All rights reserved. Keywords: Cometabolism; 4-chlorophenol; Acclimation; Inhibition; Biodegradation

1. Introduction The development of human industrial and agricultural activities leads to the synthesis of new organic compounds known as xenobiotics (Lora et al., 2000). Chlorophenols, being one such chemical, are introduced into the environment through the discharge of wastewaters originating mainly from chlorophenol production and pulping industries. They also show up in disinfected drinking water. The reported levels of chlorophenols in contaminated environments range from 150 mg/L (Valo et al., 1990) to 100–200 mg/L (Ettala et al., 1992). Many forms of chlorinated aromatics or chlorinated polyaromatics are widely utilized to control microbial contamination and degraCorresponding author. Fax: +90 312 2101260.

E-mail address: [email protected] (F.B. Dilek). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.11.005

dation. It is, therefore, not surprising that these compounds are very inhibitory to bioremediation (Hill et al., 1996; Valo et al., 1990). Therefore, their discharge into the environment is of great concern because of their toxicity and suspected carcinogenicity. Despite the recalcitrant nature of chlorophenols, there are still some efforts being made toward their biological treatment with specialized culture conditions, because of economical reasons and a low possibility of by-product formation. The microorganisms used are usually aerobes, including Pseudomonas spp., Alcaligenes spp., Azotobacter spp., Rhodococcus spp., Phanerochaere spp., and Cryptococcus spp. Aerobes are more efficient at degrading toxic compounds because they grow faster than anaerobes and usually achieve complete mineralization of toxic organic compounds, rather than transformation, as in the case of anaerobic treatment (Kim et al., 2002). However, it has been reported that

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chlorinated solvents generally cannot serve as a carbon and energy source for microbial growth, but rather must be biodegraded by cometabolism (Wang and Loh, 1999, 2000; Bali and S- engu¨l, 2002). In most of the studies with special strains of microorganisms, necessity of phenol supplementation to the growth medium was reported to induce enzymes required for 4-chlorophenol (4-CP) degradation (Wang and Loh, 1999, 2000; Hill et al., 1996; Lu et al., 1996; Kim and Hao, 1999; Hao et al., 2002). The main problems with the usage of special strains for chlorophenol degradation are, therefore, the possibility of contamination and phenol requirement, which may lead to additional pollution problems. Mixed cultures are particularly important when the emphasis is placed on complete mineralization of toxic organics to CO2. Many pure-culture studies have shown that toxic intermediates accumulate during biodegradation, because a single organism may not have the ability to completely mineralize the xenobiotic (Buitron and Gonzalez, 1996). Therefore, the treatment of chlorophenols using an activated sludge process in which a mixed culture is in action in the absence of a special growth substrate would be more meaningful, informative, and practical. The main advantage achieved by the microbial consortium formed by activated sludge is the interaction between all the species present in the flocks. Also, it is well known that the capacities of an activated sludge system can be enhanced by acclimation (Buitron et al., 1998; Kim et al., 2002). Buitron et al. (1998) reported that acclimated activated sludge degraded the chlorophenol mixture by 1 to 2 orders of magnitude faster than pure strains obtained from the acclimated consortium. Knowledge of microbial growth and substrate utilization kinetics is important for the accurate prediction of effluent quality from engineered treatment processes (Ellis et al., 1996a, b; Ellis and Anselm, 1999; Grady et al., 1996). Accurate kinetic parameters also help operation engineers to optimize operational conditions to meet discharge requirements (Ellis and Anselm, 1999). The Haldane equation is one of the most commonly used models to describe the self-inhibitory effect of a compound on its own transformation (Hao et al., 2002; Ellis et al., 1996b). In recent years great effort has been put into the modeling of the biodegradation of phenol (Monteiro et al., 2000; Kim and Hao, 1999; Hao et al., 2002) and cometabolic degradation of 4-CP in the presence of phenol as a growth substrate (Hill et al., 1996; Sae´z and Rittmann, 1993; Kim and Hao, 1999; Hao et al., 2002; Wang and Loh, 2001). Most of these studies were carried out using pure cultures under sterilized conditions. Therefore, the observed kinetic parameters cannot be applicable to model a full-scale application, in which mixed culture is responsible for substrate utilization. Another important

point is that the growth conditions of biomass used for batch degradation assay can greatly influence the observed kinetic parameters, even with pure cultures (Ellis et al., 1996a, b; Ellis and Anselm, 1999; Grady et al., 1996). Thus, the kinetic parameters for a particular culture and substrate are not necessarily constant and may reflect previous growth conditions (Ellis et al., 1996b). In this context, a parent bioreactor, which is used for the biomass source for batch experiments, should be operated within the limits of real full-scale treatment processes. Although there seems to have been a lot of study of 4-CP degradation using specialized strains growing on a specific substrate, considering that the abovementioned problems make them unfeasible for full application, it would not be wrong to state that there is still a need for further investigation of the cometabolic degradation of 4-CP facilitated solely by a nontoxic, conventional carbon source with particular emphasis on acclimation of mixed culture. Also, further investigation of the kinetics for additional understanding of the system, as well as to rationalize process design and optimize operating parameters, is needed. Therefore, in this study, we aimed to investigate the metabolic and cometabolic degradation of 4-CP using acclimated and unacclimated activated sludge cultures with an emphasis on the evaluation of biokinetic constants.

2. Materials and methods 2.1. Culture Culture was obtained from a fed-batch reactor receiving a synthetic wastewater devoid of chlorophenol and used as the initial inoculum for the batch experiments conducted with unacclimated culture. The sludge retention time (SRT) and biomass concentration in this reactor were 8 days and 2000 mg/L as mixed liquor volatile suspended solids (MLVSS), respectively. Acclimated culture was obtained from a fed-batch reactor (SRT 8 days) in which the 4-CP concentration was increased in small stepwise increments within 5 months to allow the culture to acclimatize ultimately to 130 mg 4-CP/L. Similarly, culture acclimated to 75 mg/L 2,4-dichlorophenol (2,4-DCP) was obtained from another fed-batch reactor fed with a synthetic wastewater containing 2,4-DCP. All fed-batch reactors were operated at 25 1C and aeration was provided so as to have at least 2 mg/L oxygen. Activated sludge cultures used as initial inocula for the fed-batch reactors were obtained from the activated sludge unit of the Greater Municipality of Ankara Domestic Wastewater Treatment Plant.

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2.2. Culture medium

Table 1 Composition of the synthetic wastewater (Dilek et al., 1998)

The composition of the synthetic wastewater used during the experiments is presented in Table 1. Proteose–peptone (Oxoid) was used as nitrogen and carbon source during the experiments in which the degradation of 4-CP was sought in the presence of a primary substrate. In the experiments in which 4-CP was used as the sole carbon source, proteose–peptone was excluded from the medium and NH4Cl was used to supply nitrogen to the medium. The phosphate salts were added to the synthetic medium as the source of phosphorus to the microorganisms as well as to provide a buffer capacity.

Constituent

Concentration (mg/L)

Proteose–peptone NaCl Na2SO4 K2HPO4 MgCl2  6H2O FeCl2  2H2O CaCl2  2H2O MnSO4 ZnSO4 CoSO4 CuSO4

470 (COD ¼ 500 mg/L) 407.4 44.6 44.6 3.7 3.7 3.7 0.057 0.046 0.049 0.076

2.3. Experiments Batch experiments were conducted in 500-ml Erlenmeyer flasks stoppered with cotton plugs. The working liquid volume was 250 ml. All experiments were carried out in an orbital shaking incubator set at 200 rpm and 25 1C. A stock solution of 4-CP (Merck Chemical Co., Germany) dissolved in 0.01 N NaOH was used to adjust different concentrations of 4-CP in duplicate reactors. Baseline reactors, which did not receive any 4-CP, were provided for both acclimated and unacclimated cultures as biomass control reactors. Also, 4-CP-control reactors (including 4-CP and growth medium, but not biomass) were operated under the same conditions as other reactors to follow 4-CP removal via volatilization, if any. Following the centrifugation of the effluent from batch reactors, 4-CP on biomass was extracted using 0.1 N NaOH to evaluate the degree of removal via adsorption. In the experiments in which peptone was used as the primary substrate, samples taken from the reactors at various time intervals during incubation were analyzed for biomass using optical density (OD), chemical oxygen demand (COD), and 2-hydroxy-5-chloromuconic semialdehyde (CHMS) and 4-CP concentration. Prior to CHMS, COD, and 4-CP analyses, samples were centrifuged and supernatants were used. Specific growth rate (m) values were calculated using the exponential growth phase data according to the formula lnðX =X 0 Þ ¼ m t; where X 0 and X indicate the biomass level at the beginning and at time t; respectively. MLVSS measurements were also carried out at the end of the exponential growth phase to calculate yield coefficient (Y ) and q (specific substrate utilization rate). The Y values were computed as Y ¼ ðX max  X 0 Þ=ðS0  Smin Þ; where X 0 and X max are biomass concentration as MLVSS at the start of the experiment and maximum biomass concentration reached throughout the experiments. Similarly, S 0 and Smin are corresponding substrate concentrations

as COD (Sae´z and Rittmann, 1993). In the experiments in which 4-CP was used as the sole carbon and energy source, samples were drawn for the analysis of CHMS and 4-CP only. The percentage inhibition on any biokinetic parameter was calculated using the formula percentage inhibition ¼ ((A1A2)/A1)  100, where A1 and A2 are the parameter values observed before and after 4-CP addition, respectively. 2.4. Analytical techniques Biomass growth in the batch reactors was monitored with OD measurements at 550 nm using a Bausch & Lomb Spectronic 20 spectrophotometer. CHMS concentration, the meta-cleavage product of 4-chlorocatechol, was followed by measuring OD at 380 nm (Farrell and Quilty, 1999). MLVSS and COD analyses were carried out according to standard methods (American Public Health Association, 1995). For the measurement of 4-CP concentration, two methods were concomitantly used, namely the direct photometric method (DPM) (American Public Health Association, 1995) and a highperformance liquid chromatography (HPLC) method. In the cases in which a substantial decrease was observed in 4-CP level via DPM, samples were injected onto the HPLC apparatus (Shimadzu, LC-10AT) to see the extent of removal and the formation of intermediate products, if any. The HPLC system used was equipped with a Nucleosil C18 column (4.6  250 mm), LC10Atvp solvent delivery module, SC/L0Avp system controller, and SPD-10Avp UV–Vis detector set at 280 nm. Retention time of 4-CP was 6.67 min. Solvent used in the analyses was methanol (60%), pure water (38%), and acetic acid (2%) at a flow rate of 1 ml/min (Haggblom and Young, 1990). The sample injection volume was 20 ml. All the experiments and measurements were done in duplicate and arithmetic averages were taken throughout the data analysis and calculations. Coefficients of variation for COD and MLVSS measurements were less

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than 10%, whereas they were less than 5% for 4-CP measurements.

3. Results and discussion 3.1. Toxicity and cometabolic degradation of 4-CP In this set of experiments, our aim was to observe intrinsic degradation parameters, which represent the maximum capability of the members of the microbial community with the fastest growth kinetics. The observed kinetic parameters under such conditions may help us understand ultimate reactor performance under different growth conditions (Grady et al., 1996). In the experiments, the initial biomass concentration was around 25 mg/L and COD/biomass ratio ranged from 15 to 30 on COD basis. It was reported that in the determination of intrinsic growth-associated kinetics parameters, the initial value of substrate to biomass should be around 20 on COD basis. Another important point to be emphasized is that in the case of mixed cultures, the manner in which the culture has been developed determines the type of species, which may affect the kinetics of the mixed cultures (Grady et al., 1996). Therefore, in our study, SRT of the parent bioreactor was selected as 8 days to represent a real fullscale application. Also, biomass for batch tests was drawn when the parent reactor was in steady-state operation to supply biomass with constant characteristics for different batch assays (Sae´z and Rittmann, 1993). Time-course variations in biomass as OD, COD, and 4-CP were followed at various initial concentrations of 4-CP for both unacclimated and acclimated cultures. In Fig. 1, variations in biomass concentration as OD and COD for 200 mg/L of 4-CP are presented for acclimated culture. Similar trends for OD and COD were also observed for other concentrations (data were not given). The removal of 4-CP via evaporation and adsorption on biomass was negligible (o5%) under the studied conditions. It was observed that the lag phase (between

Fig. 1. Time-course variations in COD and OD values at 200 mg/L 4CP for the acclimated culture.

5 and 10 h depending on the initial 4-CP concentration) for the unacclimated culture disappeared when the culture was acclimated to 4-CP. Similarly, a linear increase in the lag time was observed in the study of Hill et al. (1996), in which cometabolic degradation of 4-CP by Alcaligenes eutrophus was investigated, but the effect of acclimation on lag-phase duration was not investigated. Time required to reach the stationary phase was also variable depending the 4-CP concentration for both unacclimated and acclimated cultures (Table 2). The time required to reach stationary phase for acclimated culture was observed to increase exponentially with increasing 4-CP concentration (r2 ¼ 0:986). The stationary phase, for the acclimated culture, was nearly reached at the end of 1 day (Table 2) when the 4-CP concentration was below 200 mg/L. However, this period was observed to extend up to 2 days (Table 2) when the 4-CP concentration was increased to 300 mg/ L, possibly due to the toxicity exerted by 4-CP at high concentrations. Therefore, it can be suggested that although mixed culture degraded peptone and 4-CP simultaneously, the degradation rate of peptone was determined by 4-CP. Also, it is important to note that the growth of acclimated activated sludge was possible even at a high initial 4-CP concentration of 300 mg/L. In comparison, Hill et al. (1996) reported that growth of A. eutrophus was completely inhibited by 4-CP beyond the concentration of 69 mg/L in the presence of 1080 mg/L phenol. Also, Wang and Loh (1999) reported that when initial 4-CP concentration was increased to 300 mg/L, cells could not grow on glucose even after an extended period of incubation. Hence, better growth ability of our culture is thought to be due to the selection of microorganisms tolerant to 4-CP during the acclimation period. The effects of 4-CP on m; COD removal efficiency, q; and Y are shown in Table 2 for both unacclimated and acclimated cultures. As can be seen from Table 2, the m values decreased with increasing concentrations of 4-CP for both cultures. The percentage inhibition observed on m (the percentage decrease in m value compared to that of the baseline reactor) for both cultures is shown in Fig. 2. The value of IC50 (concentration causing 50% inhibition) on the basis of m was found to be 218 mg/L for 4-CP-acclimated culture, whereas it was 130 mg/L when an unacclimated culture was used. Although a remarkable decrease in the toxic effect of 4-CP was observed after acclimation, the m values of the unacclimated culture were higher than those of the acclimated culture (Table 2). This was also apparent from the m values of acclimated and unacclimated cultures receiving no 4-CP (Table 2). Similar to the observed m values, the qðm=Y Þ values of unacclimated culture were much higher compared to those of acclimated culture. These results can also be concluded from the time required to reach the stationary phase of growth, as a

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Table 2 Batch experiment biokinetic coefficients at various concentrations of 4-CP for both cultures 4-CP (mg/L)

ta (h)

%COD removal

Y (mg MLVSS/mg COD)

4-CP-acclimated culture

0 130 200 300 390

11 25 28 49 76

6271 7478 6771 7277 2471

0.7370.15 0.5370.01 0.5770.02 0.4070.08 0.5970.02

Unacclimated culture

0 57 112 155 274

11 13 22 23 23

6472 6475 4671 3571 4072

0.4770.10 0.5270.08 0.5270.03 0.670.03 0.470.02

a

q (mg COD/(mg MLVSS h)) 0.17970.1 0.13770.08 0.11270.07 0.10570.05 0.03370.007 0.780570.1 0.49270.1 0.434670.07 0.25670.09 0.26570.03

m (1/h) 0.13170.011 0.07370.007 0.07070.007 0.04270.004 0.02070.003 0.36770.064 0.25670.007 0.22670.004 0.15470.008 0.10670.002

Time required to reach stationary phase.

Fig. 2. The percentage inhibition of m caused by 4-CP.

much longer time was required in the case of acclimated culture due to slower growth and substrate utilization ability. A variety of phenomena have been proposed to explain the acclimation phase; one is the selection and multiplication of specialized microorganisms during the acclimation phase (Wiggins et al., 1987; Rittmann and McCarty, 2001). The results of our study suggest that 4-CP-tolerant-microorganims, having slower growth ability, were selected. In support of this, Buitron et al. (1998) claimed that acclimated activated sludge was composed of some bacteria having low growth-rate coefficients, but their participation was essential for efficient biodegradation of chlorophenols. For the acclimated culture, the COD removal efficiencies achieved for the studied range of 4-CP (130–300 mg/L) were almost the same and slightly higher than that observed for the baseline reactor of the acclimated culture (Table 2). Hence, it can be said that culture acclimated to 130 mg 4-CP/L (which corresponds to the IC50 value on the basis of m depression for unacclimated culture) was not adversely affected by 4-CP up to 300 mg/L on the basis of COD removal efficiency. On the other hand, in the case of the unacclimated culture, the baseline COD removal efficiency decreased from 64 to 46 and 35% when 4-CP concentration was increased to 112 and 155 mg/L,

respectively (Table 2). When toxicity of 4-CP is compared to that of 2,4-DCP, it can be said that 2,4DCP is more toxic than 4-CP, as the presence of 2,4DCP resulted in lower COD removal efficiency (Sahinkaya and Dilek, 2002). Similarly, Buitron et al. (1998) reported that degradation of 4-CP by acclimated activated sludge is faster compared to that of 2,4DCP, possibly due to a more toxic effect of 2,4-DCP on microorganisms. As seen from Table 2, the Y value of 0.73 mg MLVSS/mg COD for the baseline case of acclimated culture was surprisingly high. The reason for obtaining such a high Y value may be attributed to the fact that energy gained from substrate utilization was channeled into biomass growth rather than maintenance when 4CP was excluded from the medium. When 4-CP was added to the growth medium at different initial concentrations, the Y values decreased to between 0.40 and 0.59 mg MLVSS/mg COD for the acclimated culture. In the case of unacclimated culture, the Y value in the absence of 4-CP was 0.47 mg MLVSS/mg COD and it ranged between 0.4 and 0.6 mg MLVSS/mg COD in the presence of 4-CP at different concentrations (Table 2). Therefore, these observations led us to conclude that for both cultures, Y values did not correlate to the initial 4-CP concentration. Also, it was observed that in the presence of 4-CP the average Y value was observed to be 0.5270.085 and 0.5170.082 mg MLVSS/mg COD for acclimated and unacclimated culture, respectively. Therefore, for both cultures Y values were almost constant in the presence of 4-CP. Similarly, Sae´z and Rittmann (1993) reported that the Y value for phenol was not affected by the presence of 4-CP. They attributed the reason for this observation to the fact that the biomass was shunting about 0.5% of the electrons gained by phenol oxidation to regeneration of NADPH for 4-CP transformation, and this very low diversion of electrons did not have any significant effect on Y : In contrast, Hill et al. (1996) found that the total biomass yield of A. eutrophus was

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dropped drastically when 4-CP was present in the growth medium. They observed that the biomass yield decreased to 0.36 in the presence of 60 ppm 4-CP compared to 0.60 when phenol was present as the sole source of carbon. Compared with the 2,4-DCP case conducted earlier (Sahinkaya and Dilek, 2002), the Y values decreased with increasing 2,4-DCP for the acclimated culture (Sahinkaya and Dilek, 2002), hence, a certain effect of 2,4-DCP concentration variation on the biomass yield was obvious, unlike 4-CP. In regard to the removal of 4-CP, no removal was detected within 30 h in reactors inoculated with the unacclimated culture (data were not shown). However, when the acclimated culture was used, nearly complete removal was observed within 24 and 48 h for 130 and 200 mg 4-CP/L, respectively (Fig. 3). Hence, results suggest that degradation of 4-CP by activated sludge requires acclimation of biomass. This phenomenon was also reported in the studies of Buitron and Gonzalez (1996) and Buitron et al. (1998), for the degradation of 4-CP, and Sahinkaya and Dilek (2002) for the degradation of 2,4-DCP. Although 80% removal of 200 mg 4-CP/L could be achieved within 1 day, no remarkable degradation of 300 mg 4-CP/L was observed within 1 day. However, following the first day of incubation, 4-CP removal rate increased sharply and almost complete removal of 300 mg/L was achieved at the end of the second day. This can be attributed to the fact that the culture had been acclimated to 130 mg 4-CP/L in the fed-batch reactor; therefore, a longer time was required for adaptation to the higher 4-CP concentrations. Furthermore, 4-CP degradation was not observed at all within 76 h when the initial 4-CP concentration was 390 mg/L. This observation indicates that culture acclimated to 130 mg 4-CP/L could degrade 4-CP up to 300 mg/L; hence acclimation to higher 4-CP concentrations seems to be required in order to remove 4-CP concentrations higher than 300 mg/L. As can be inferred from Fig. 3, the removal of 4-CP occurs in two phases, first a slow and then a rapid or

accelerated removal phase. The specific degradation rates (SDR) of 4-CP at the accelerated phases were calculated for each initial 4-CP concentration and Fig. 4 was prepared to show the relation between them. This figure shows that SDR of 4-CP remained nearly constant up to 300 mg/L and decreased sharply when the 4-CP concentration was increased further to 390 mg/ L. As concerns the magnitudes, in our study, very high SDRs of 4-CP (102–717 mg 4-CP/(g MLVSS day) were observed when the acclimated culture was used. These results are in good agreement with those of Buitron et al. (1998), in which the SDR of 4-CP for acclimated activated sludge culture was found to be 784 mg/ (g MLVSS day), which is much higher than the SDR of 1.8 mg chlorinated phenols/(g MLVSS day) for an unacclimated activated sludge. Similarly, Sahinkaya and Dilek (2002) had observed the removal rates of 226.5 and 129 mg 2,4-DCP/(g MLVSS day) for the initial concentrations of 76.6 and 194.5 mg/L of 2,4-DCP, respectively. In contrast, Vallecillo et al. (2000) reported very low degradation rates of 4-CP (1.2–4.2 mg 4-CP/ (g MLVSS day) and 2,4-DCP (1.2–1.7 mg 2,4-DCP/ (g MLVSS day), with no differences in the toxic elimination rate between first and second feed, which could be taken as an indication of ‘‘no effect of acclimation’’ in their study. Observed SDR values for acclimated activated sludge are also much higher compared to those for pure strains of other studies. Buitron et al. (1998) reported that SDR of 4-CP for Chryseomonas luteola, Aeromonas sp., Pseudomonas sp., and Flavimonas oryzihabitans were 39, 47, 37, and 44 mg/ (g MLVSS day) compared to 784 mg/(g MLVSS day) for acclimated activated sludge. In the study of Wang and Loh (1999), when the medium pH was kept between 6.5 and 7.5, the degradation rates of 4-CP at initial concentrations of 100 and 200 mg/L by Pseudomonas putida were observed as 9–11 and 10 mg/(L h), respectively, in the presence of glucose as a growth substrate. On the other hand, when pH of the medium was not regulated, the pH quickly dipped below 4.5, consequently stopping the further transformation of 4-CP, and the average transformation rate of 4-CP at an initial

Fig. 3. Variations in 4-CP concentration with time for the acclimated culture in the presence of peptone.

Fig. 4. Specific degradation rate (SDR) of 4-CP for the acclimated culture at various concentrations of 4-CP.

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concentration of 200 mg/L was only about 3 mg/(L h). In our study the pH of the medium was between 7 and 8 throughout the study, and the degradation rates of 4-CP were observed as 8.15, 10.15, 10.22, and 1.03 mg/(L h) for initial concentrations of 130, 200, 300, and 390 mg/L, respectively. Therefore, our findings are in good agreement with the literature findings. Based on the above results, it is apparent that the increase in 4-CP from 130 to 300 mg/L has no significant effect on the average 4-CP transformation rate. In another study, Lu et al. (1996) reported that the initial 4-CP removal rates by Pseudomonas testosterone, Aeromonas radiobacter, P. putida, and Pseudomonas aeruginosa were 10, 1.2, 1.1, and 2.9 mg/(L day) even at 10 mg/L. Therefore, the studied concentrations and degradation rates are very low compared to those in our study. In order to increase 4-CP removal efficiency and rate, Buitron et al. (1998) used a mixed culture formed by selected pure strains having the ability to degrade 4-CP and they observed that the biodegradation capacity of the mixture of isolates was much lower than for the acclimated activated sludge. Therefore, a good biodegradation system requires the maintenance of a wide range of bacterial species at significant concentrations in order to adapt to transient fluctuations in concentrations or types of cometabolites that may become present in industrial effluents (Buitron et al., 1998). Further comparison of this study with the literature reports revealed that in contrast to the findings of Wang and Loh (2000), Kim and Hao (1999), and Hill et al. (1996), which pointed to the necessity of phenol supplementation as the primary substrate for the degradation of 4-CP, cometabolic degradation of 4-CP is possible using the acclimated culture in the absence of phenol. Thus, in our study, it was shown that a nontoxic organic compound, peptone, can be used during the cometabolic degradation of 4-CP and the use of phenol, which is also a toxic compound, could be avoided. There are some supportive literature reports on the disadvantage of using phenol as the primary substrate (Wang and Loh, 1999; Bali and S- engu¨l, 2002). In the case of using conventional growth substrate, the cometabolic enzymes required for 4-CP transformation were most probably induced by 4-CP; cofactor NADH required for 4-CP transformation could be efficiently formed through the oxidation of conventional growth substrate (Wang and Loh, 1999). Hence, it can be inferred that, in our study, after acclimation, the utilization rate of peptone increased and NADH was quickly regenerated, consequently facilitating the transformation of 4-CP.

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substrate was also investigated since the addition of a primary substrate would result in a substantial increase in the overall treatment process cost. Therefore, it is worth devoting attention to the degradation of 4-CP when it is present as the sole carbon and energy source. In this set of experiments, in addition to an unacclimated and a 4-CP-acclimated culture, a culture previously acclimated to 2,4-DCP was used. In the latter case, 4-CP was again the only carbon and energy source (i.e., no 2,4-DCP was present). In this set of experiments, initial biomass concentration was around 50 mg/L. Time-course variations of 4-CP are given in Figs. 5 and 6, for the acclimated and the unacclimated culture, respectively. Fig. 5 shows that 50 and 140 mg 4-CP/L could be removed almost completely within 1 and 3 days, respectively, without any lag phase when 4-CPacclimated culture was used. Although 200 mg 4-CP/L could also be removed completely within 3 days, the initial removal rate of 4-CP was lower. On the other hand, the use of 2,4-DCP-acclimated culture for the degradation of 138 mg 4-CP/L resulted in complete removal within 5 days. About 3 days of lag period was required for 2,4-DCP-acclimated culture, whereas no lag phase was required when 4-CP-acclimated culture was used. When the unacclimated culture was used, 4-CP removals achieved were only 44% and 30% for the initial 4-CP concentrations of 26 and 130 mg/L, respectively, at the end of 18 days (Fig. 6). Therefore, it can be stated that 2,4-DCP-acclimated culture exhibited less ability than 4-CP-acclimated culture, but better ability than the unacclimated culture, toward the removal of 4-CP. The ability of 4-CP-acclimated culture to use 2,4-DCP was also shown by Sahinkaya and Dilek (2002). Similarly, Lu et al. (1996) reported that if a species can effectively metabolize one type of chlorophenols, it is reasonable to expect that this species also can effectively utilize other structurally analogous chlorophenols.

3.2. Treatment of 4-CP when present as the sole organic carbon source In addition to the experimental studies discussed above, degradation of 4-CP in the absence of primary

Fig. 5. 4-CP concentrations with time for reactors in which 4-CP served as the sole organic carbon source for the acclimated culture (initial cell concentration was 50 mg/L).

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Fig. 6. 4-CP concentrations with time for reactors in which 4-CP served as the sole organic carbon source for the unacclimated culture.

The maximum degradation rates of 4-CP by the 4-CPacclimated culture in absence of peptone were found to be 2.56, 1.93, and 2.9 mg/(L h) for initial 4-CP concentrations of 50, 140, and 200 mg/L, respectively, compared to the maximum degradation rates of 8.15–10.22 mg/(L h) for initial 4-CP concentration of between 130 and 300 mg/L by acclimated culture in the presence of peptone. The maximum 4-CP degradation rate for 130 mg/L by an unacclimated culture in the absence of peptone was 0.53 mg/(L h), which is 4 and 15 times lower than that by the acclimated culture in the absence and the presence of peptone, respectively. The observed higher degradation rate in the presence of peptone was due to increased biomass concentration because peptone was responsible only for biomass production and did not cause any increase in SDR of 4-CP, which is discussed later.

concentrations. As can be seen from the figure, complete removal of 4-CP was achieved for all concentrations studied. Also, it was observed that the time required to see complete degradation increased exponentially (r2 ¼ 0:97) with increasing 4-CP concentrations (data not shown). Compared to Fig. 5, it can be clearly concluded that increased initial concentration of biomass caused an increase in degradation rate of 4-CP. For example, at an initial concentration of about 140 mg/L 4-CP, around 3 days was required to achieve complete degradation when initial biomass concentration was around 50 mg/L, whereas around 1 day was required to completely remove 160 mg/L 4-CP when initial biomass concentration was around 200 mg/L. Similarly, the degradation rate of 4-CP increased from 1.93 (for 140 mg 4-CP/L) to around 4.5 mg/(L h) (for 160 mg 4-CP/L) with increasing initial biomass concentration. Fig. 7 also shows the relation between specific degradation rates and initial 4-CP concentrations. The decreased 4-CP degradation rate with increasing initial 4-CP concentration implies that 4-CP acts as an inhibitor. Therefore, the Haldane substrate inhibition was used to model the inhibitory effect of 4-CP on its own transformation. Application of experimental data to the Haldane equation gave excellent fit to our experimental data (Fig. 7) as r2 was observed to be 0.986. The least-square error method with the help of Mathlab 6.5 was used to obtain kinetic parameters. The

3.3. Development of kinetic model Degradation of 4-CP in the absence of peptone was also investigated at higher initial biomass concentrations to investigate the effect of initial biomass concentration and obtain kinetic parameters. In this set of experiments, initial biomass concentration was kept high to accelerate degradation rate of 4-CP and avoid further acclimation of culture at longer lag-phase durations, which may affect observed biokinetic parameters. Adaptation of cells at prolonged lag phase during phenol degradation was also reported in the study of Sae´z and Rittmann (1993). They reported that acclimation of cells to high concentrations of phenol during the assay caused some change in kinetic parameters due to a reduced inhibitory effect. In this context, in our experimental design, in order to avoid so much change in culture history and correctly estimate kinetic parameters, degradation experiments were carried out at an initial biomass concentration of 200710 mg/L. Therefore, 4-CP/biomass ratios ranged between 0.05 and 0.94 on COD basis. Fig. 7 shows time-course variation in 4-CP concentration with time and SDR at various initial 4-CP

Fig. 7. Time-course variations in 4-CP and dependence of SDR on initial 4-CP concentration for acclimated culture (initial cell concentration was 200 mg/L).

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Haldane parameters for mixed culture acclimated to 4CP were obtained as qm ¼ 41:17 mg 4-CP/(g MLVSS h), K s ¼ 1:104 mg=L; and K i ¼ 194:4 mg=L: Therefore, the observed kinetic equation is dðSÞ 41:17 S X ¼ dðtÞ 1:104 þ S þ

S2 194:4

:

(1)

Wang and Loh (2001) reported that in 4-CP degradation, addition of growth substrate, which is structurally dissimilar to 4-CP, does not affect specific degradation of 4-CP and it is responsible only for more cell production. In this context, the validity of our kinetic model for 4-CP degradation was checked by applying the observed model to one set of experiments in which peptone (500 mg/L COD) was available as the primary growth substrate. In this set of experiments, initial biomass and 4-CP concentrations were 25 and 130 mg/L, respectively. Change in biomass concentration was modeled using a logistic growth equation (Shuler and Kargi, 1992). The equation is   dX X ¼ kX 1  ; (2) dt K where k and K are growth-associated constant and carrying capacity of the system, respectively. The integration of Eq. (2) yields the logistic curve X¼

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removed (data not shown). Also, the maximum absorbance value showed an increasing trend with increasing initial 4-CP concentration. After observation of the peak value, a sharp decrease in absorbance to its original value indicated complete degradation of 4-CP via the meta-cleavage pathway. HPLC results of influent and effluent samples also indicated complete removal of 4-CP without any by-product observation. As an example, Fig. 9 shows HPLC diagrams of one selected batch experiment. The results of this study indicated that the toxic effects of 4-CP decreased remarkably on the basis of m and COD removal efficiency, after acclimation. It was shown that in the cometabolic degradation of 4-CP, a nontoxic organic compound, peptone, can be used instead of phenol. Although 4-CP removal by an unacclimated culture was negligible, efficient removal via the meta-cleavage pathway by the acclimated culture both in the presence and in the absence of peptone was observed. The Haldane equation seems to be an adequate expression as 4-CP inhibits its own transformation at high concentrations. It was observed that peptone did not affect the specific degradation rate of 4-CP and it was responsible only for more cell production. Also, 4-CP could be degraded by 2,4DCP-acclimated culture, although not as effectively as by 4-CP-acclimated culture.

X 0 ekt ; 1  XK0 ð1  ekt Þ

where X 0 represents initial biomass concentration. Experimental results gave the carrying capacity as 282.6 mg/L and k was determined to be 0.25227 0.03 h1 (r2 ¼ 0:953) with the help of Mathlab 6.5. Therefore, with the simultaneous solution of Eqs. (1) and (2) using the obtained kinetic parameters, timecourse variations in 4-CP and biomass can be predicted (Fig. 8). Observed differential equations were numerically solved using POLYMATH 4.02. The program used the Runge–Kutta–Fehlberg numerical integration routine. Change in biomass measured by OD was converted to mg/L MLVSS using the linear relation. It can be seen from the figure that time-course variations in both biomass and 4-CP were reasonably predicted. Therefore, similar to results of Wang and Loh (2001), the presence of peptone did not increase the specific degradation rate of 4-CP and it was responsible only for the production of biomass. During the batch degradation studies, a yellowish color accumulated during the degradation of 4-CP due to production of CHMS, indicating meta cleavage of 4-CP (Sae´z and Rittmann, 1991; Wang and Loh, 1999; Farrell and Quilty, 1999). The color of the filtered medium was measured at 380 nm and it was observed that the intermediate concentration reached its maximum value when 4-CP was just about completely

Fig. 8. Simultaneous modeling of 4-CP transformation and biomass growth in the presence of peptone for acclimated culture.

Fig. 9. HPLC diagram of influent and effluent of batch experiments inoculated with acclimated culture receiving 200 mg 4-CP/L.

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