biodegradation of 4-chlorophenol by acclimated and unacclimated activated sludgeevaluation of...

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Environmental Research 99 (2005) 243–252

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Biodegradation of 4-chlorophenol by acclimated and unacclimatedactivated 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 218mg/L with the acclimation of the culture. Although an increase in 4-CP concentration up to 300mg/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 300mg/L. The Haldane kinetic model adequately predicted the biodegradation of 4-CP

and the kinetic constants obtained were qm ¼ 41:17mg=ðgMLVSShÞ; K s ¼ 1:104mg=L; and K i ¼ 194:4mg=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 agricultur-al activities leads to the synthesis of new organiccompounds known as xenobiotics (Lora et al., 2000).Chlorophenols, being one such chemical, are introducedinto the environment through the discharge of waste-waters originating mainly from chlorophenol produc-tion and pulping industries. They also show up indisinfected drinking water. The reported levels ofchlorophenols in contaminated environments rangefrom 150 mg/L (Valo et al., 1990) to 100–200mg/L(Ettala et al., 1992). Many forms of chlorinatedaromatics or chlorinated polyaromatics are widelyutilized to control microbial contamination and degra-

e front matter r 2004 Elsevier Inc. All rights reserved.

vres.2004.11.005

ing author. Fax: +90312 2101260.

ess: [email protected] (F.B. Dilek).

dation. It is, therefore, not surprising that thesecompounds are very inhibitory to bioremediation (Hillet al., 1996; Valo et al., 1990). Therefore, their dischargeinto the environment is of great concern because of theirtoxicity and suspected carcinogenicity.

Despite the recalcitrant nature of chlorophenols, thereare still some efforts being made toward their biologicaltreatment with specialized culture conditions, because ofeconomical reasons and a low possibility of by-productformation. The microorganisms used are usually aero-bes, including Pseudomonas spp., Alcaligenes spp.,Azotobacter spp., Rhodococcus spp., Phanerochaere

spp., and Cryptococcus spp. Aerobes are more efficientat degrading toxic compounds because they grow fasterthan anaerobes and usually achieve complete miner-alization of toxic organic compounds, rather thantransformation, as in the case of anaerobic treatment(Kim et al., 2002). However, it has been reported that

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ARTICLE IN PRESSE. Sahinkaya, F.B. Dilek / Environmental Research 99 (2005) 243–252244

chlorinated solvents generally cannot serve as a carbonand energy source for microbial growth, but rather mustbe biodegraded by cometabolism (Wang and Loh, 1999,2000; Bali and S-engul, 2002).

In most of the studies with special strains ofmicroorganisms, necessity of phenol supplementationto the growth medium was reported to induce enzymesrequired for 4-chlorophenol (4-CP) degradation (Wangand Loh, 1999, 2000; Hill et al., 1996; Lu et al., 1996;Kim and Hao, 1999; Hao et al., 2002). The mainproblems with the usage of special strains for chlor-ophenol degradation are, therefore, the possibility ofcontamination and phenol requirement, which may leadto additional pollution problems.

Mixed cultures are particularly important when theemphasis is placed on complete mineralization of toxicorganics to CO2. Many pure-culture studies have shownthat toxic intermediates accumulate during biodegrada-tion, because a single organism may not have the abilityto completely mineralize the xenobiotic (Buitron andGonzalez, 1996). Therefore, the treatment of chloro-phenols using an activated sludge process in which amixed culture is in action in the absence of a specialgrowth substrate would be more meaningful, informa-tive, and practical. The main advantage achieved by themicrobial consortium formed by activated sludge is theinteraction between all the species present in the flocks.Also, it is well known that the capacities of an activatedsludge system can be enhanced by acclimation (Buitronet al., 1998; Kim et al., 2002). Buitron et al. (1998)reported that acclimated activated sludge degraded thechlorophenol mixture by 1 to 2 orders of magnitudefaster than pure strains obtained from the acclimatedconsortium.

Knowledge of microbial growth and substrate utiliza-tion kinetics is important for the accurate prediction ofeffluent quality from engineered treatment processes(Ellis et al., 1996a, b; Ellis and Anselm, 1999; Gradyet al., 1996). Accurate kinetic parameters also helpoperation engineers to optimize operational conditionsto meet discharge requirements (Ellis and Anselm,1999). The Haldane equation is one of the mostcommonly used models to describe the self-inhibitoryeffect of a compound on its own transformation (Haoet al., 2002; Ellis et al., 1996b). In recent years greateffort has been put into the modeling of the biodegrada-tion of phenol (Monteiro et al., 2000; Kim and Hao,1999; Hao et al., 2002) and cometabolic degradation of4-CP in the presence of phenol as a growth substrate(Hill et al., 1996; Saez and Rittmann, 1993; Kim andHao, 1999; Hao et al., 2002; Wang and Loh, 2001).Most of these studies were carried out using purecultures under sterilized conditions. Therefore, theobserved kinetic parameters cannot be applicable tomodel a full-scale application, in which mixed culture isresponsible for substrate utilization. Another important

point is that the growth conditions of biomass used forbatch degradation assay can greatly influence theobserved kinetic parameters, even with pure cultures(Ellis et al., 1996a, b; Ellis and Anselm, 1999; Grady etal., 1996). Thus, the kinetic parameters for a particularculture and substrate are not necessarily constant andmay reflect previous growth conditions (Ellis et al.,1996b). In this context, a parent bioreactor, which isused for the biomass source for batch experiments,should be operated within the limits of real full-scaletreatment processes.

Although there seems to have been a lot of study of4-CP degradation using specialized strains growingon a specific substrate, considering that the above-mentioned problems make them unfeasible for fullapplication, it would not be wrong to state that thereis still a need for further investigation of the cometa-bolic degradation of 4-CP facilitated solely by anontoxic, conventional carbon source with particularemphasis on acclimation of mixed culture. Also,further investigation of the kinetics for additionalunderstanding of the system, as well as to rationalizeprocess design and optimize operating parameters, isneeded. Therefore, in this study, we aimed to investigatethe metabolic and cometabolic degradation of 4-CPusing acclimated and unacclimated activated sludgecultures with an emphasis on the evaluation ofbiokinetic constants.

2. Materials and methods

2.1. Culture

Culture was obtained from a fed-batch reactorreceiving a synthetic wastewater devoid of chlorophenoland used as the initial inoculum for the batch experi-ments conducted with unacclimated culture. The sludgeretention time (SRT) and biomass concentration in thisreactor were 8 days and 2000mg/L as mixed liquorvolatile suspended solids (MLVSS), respectively.

Acclimated culture was obtained from a fed-batchreactor (SRT 8 days) in which the 4-CP concentrationwas increased in small stepwise increments within 5months to allow the culture to acclimatize ultimately to130mg 4-CP/L. Similarly, culture acclimated to 75mg/L2,4-dichlorophenol (2,4-DCP) was obtained from an-other fed-batch reactor fed with a synthetic wastewatercontaining 2,4-DCP. All fed-batch reactors were oper-ated at 25 1C and aeration was provided so as to have atleast 2mg/L oxygen.

Activated sludge cultures used as initial inocula forthe fed-batch reactors were obtained from the activatedsludge unit of the Greater Municipality of AnkaraDomestic Wastewater Treatment Plant.

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Table 1

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

Constituent Concentration (mg/L)

Proteose–peptone 470 (COD ¼ 500mg/L)

NaCl 407.4

Na2SO4 44.6

K2HPO4 44.6

MgCl2 � 6H2O 3.7

FeCl2 � 2H2O 3.7

CaCl2 � 2H2O 3.7

MnSO4 0.057

ZnSO4 0.046

CoSO4 0.049

CuSO4 0.076

E. Sahinkaya, F.B. Dilek / Environmental Research 99 (2005) 243–252 245

2.2. Culture medium

The composition of the synthetic wastewater usedduring the experiments is presented in Table 1.Proteose–peptone (Oxoid) was used as nitrogen andcarbon source during the experiments in which thedegradation of 4-CP was sought in the presence of aprimary substrate. In the experiments in which 4-CP wasused as the sole carbon source, proteose–peptone wasexcluded from the medium and NH4Cl was used tosupply nitrogen to the medium. The phosphate saltswere added to the synthetic medium as the source ofphosphorus to the microorganisms as well as to providea buffer capacity.

2.3. Experiments

Batch experiments were conducted in 500-ml Erlen-meyer flasks stoppered with cotton plugs. The workingliquid volume was 250ml. All experiments were carriedout in an orbital shaking incubator set at 200 rpmand 25 1C.

A stock solution of 4-CP (Merck Chemical Co.,Germany) dissolved in 0.01N NaOH was used to adjustdifferent concentrations of 4-CP in duplicate reactors.Baseline reactors, which did not receive any 4-CP, wereprovided for both acclimated and unacclimated culturesas biomass control reactors. Also, 4-CP-control reactors(including 4-CP and growth medium, but not biomass)were operated under the same conditions as otherreactors to follow 4-CP removal via volatilization, ifany.

Following the centrifugation of the effluent frombatch reactors, 4-CP on biomass was extracted using0.1N NaOH to evaluate the degree of removal viaadsorption.

In the experiments in which peptone was used as theprimary substrate, samples taken from the reactors atvarious time intervals during incubation were analyzedfor biomass using optical density (OD), chemical oxygendemand (COD), and 2-hydroxy-5-chloromuconic semi-aldehyde (CHMS) and 4-CP concentration. Prior toCHMS, COD, and 4-CP analyses, samples werecentrifuged and supernatants were used. Specific growthrate (m) values were calculated using the exponentialgrowth phase data according to the formula lnðX=X 0Þ ¼

m t; where X 0 and X indicate the biomass level at thebeginning and at time t; respectively. MLVSS measure-ments were also carried out at the end of the exponentialgrowth phase to calculate yield coefficient (Y ) and q

(specific substrate utilization rate). The Y values werecomputed as Y ¼ ðXmax � X 0Þ=ðS0 � SminÞ; where X 0

and Xmax are biomass concentration as MLVSS at thestart of the experiment and maximum biomass concen-tration reached throughout the experiments. Similarly,S0 and Smin are corresponding substrate concentrations

as COD (Saez and Rittmann, 1993). In the experimentsin which 4-CP was used as the sole carbon and energysource, samples were drawn for the analysis of CHMSand 4-CP only. The percentage inhibition on anybiokinetic parameter was calculated using the formulapercentage inhibition ¼ ((A1�A2)/A1)� 100, where A1

and A2 are the parameter values observed before andafter 4-CP addition, respectively.

2.4. Analytical techniques

Biomass growth in the batch reactors was monitoredwith OD measurements at 550 nm using a Bausch &Lomb Spectronic 20 spectrophotometer. CHMS con-centration, the meta-cleavage product of 4-chlorocate-chol, was followed by measuring OD at 380 nm (Farrelland Quilty, 1999). MLVSS and COD analyses werecarried out according to standard methods (AmericanPublic Health Association, 1995). For the measurementof 4-CP concentration, two methods were concomitantlyused, namely the direct photometric method (DPM)(American Public Health Association, 1995) and a high-performance liquid chromatography (HPLC) method.In the cases in which a substantial decrease wasobserved in 4-CP level via DPM, samples were injectedonto the HPLC apparatus (Shimadzu, LC-10AT) to seethe extent of removal and the formation of intermediateproducts, if any. The HPLC system used was equippedwith a Nucleosil C18 column (4.6� 250mm), LC-10Atvp solvent delivery module, SC/L0Avp systemcontroller, and SPD-10Avp UV–Vis detector set at280 nm. Retention time of 4-CP was 6.67min. Solventused in the analyses was methanol (60%), pure water(38%), and acetic acid (2%) at a flow rate of 1ml/min(Haggblom and Young, 1990). The sample injectionvolume was 20 ml.

All the experiments and measurements were done induplicate and arithmetic averages were taken through-out the data analysis and calculations. Coefficients ofvariation for COD and MLVSS measurements were less


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

3. Results and discussion

3.1. Toxicity and cometabolic degradation of 4-CP

In this set of experiments, our aim was to observeintrinsic degradation parameters, which represent themaximum capability of the members of the microbialcommunity with the fastest growth kinetics. Theobserved kinetic parameters under such conditionsmay help us understand ultimate reactor performanceunder different growth conditions (Grady et al., 1996).In the experiments, the initial biomass concentrationwas around 25mg/L and COD/biomass ratio rangedfrom 15 to 30 on COD basis. It was reported that in thedetermination of intrinsic growth-associated kineticsparameters, the initial value of substrate to biomassshould be around 20 on COD basis. Another importantpoint to be emphasized is that in the case of mixedcultures, the manner in which the culture has beendeveloped determines the type of species, which mayaffect the kinetics of the mixed cultures (Grady et al.,1996). Therefore, in our study, SRT of the parentbioreactor was selected as 8 days to represent a real full-scale application. Also, biomass for batch tests wasdrawn when the parent reactor was in steady-stateoperation to supply biomass with constant character-istics for different batch assays (Saez and Rittmann,1993).

Time-course variations in biomass as OD, COD, and4-CP were followed at various initial concentrations of4-CP for both unacclimated and acclimated cultures. InFig. 1, variations in biomass concentration as OD andCOD for 200mg/L of 4-CP are presented for acclimatedculture. Similar trends for OD and COD were alsoobserved for other concentrations (data were not given).The removal of 4-CP via evaporation and adsorption onbiomass was negligible (o5%) under the studiedconditions. It was observed that the lag phase (between

Fig. 1. Time-course variations in COD and OD values at 200mg/L 4-

CP for the acclimated culture.

5 and 10 h depending on the initial 4-CP concentration)for the unacclimated culture disappeared when theculture was acclimated to 4-CP. Similarly, a linearincrease in the lag time was observed in the study of Hillet al. (1996), in which cometabolic degradation of 4-CPby Alcaligenes eutrophus was investigated, but the effectof acclimation on lag-phase duration was not investi-gated. Time required to reach the stationary phase wasalso variable depending the 4-CP concentration for bothunacclimated and acclimated cultures (Table 2). Thetime required to reach stationary phase for acclimatedculture was observed to increase exponentially withincreasing 4-CP concentration (r2 ¼ 0:986). The sta-tionary phase, for the acclimated culture, was nearlyreached at the end of 1 day (Table 2) when the 4-CPconcentration was below 200mg/L. However, thisperiod was observed to extend up to 2 days (Table 2)when the 4-CP concentration was increased to 300mg/L, possibly due to the toxicity exerted by 4-CP at highconcentrations. Therefore, it can be suggested thatalthough mixed culture degraded peptone and 4-CPsimultaneously, the degradation rate of peptone wasdetermined by 4-CP. Also, it is important to note thatthe growth of acclimated activated sludge was possibleeven at a high initial 4-CP concentration of 300mg/L. Incomparison, Hill et al. (1996) reported that growth of A.

eutrophus was completely inhibited by 4-CP beyond theconcentration of 69mg/L in the presence of 1080mg/Lphenol. Also, Wang and Loh (1999) reported that wheninitial 4-CP concentration was increased to 300mg/L,cells could not grow on glucose even after an extendedperiod of incubation. Hence, better growth ability of ourculture is thought to be due to the selection ofmicroorganisms tolerant to 4-CP during the acclimationperiod.

The effects of 4-CP on m; COD removal efficiency, q;and Y are shown in Table 2 for both unacclimated andacclimated cultures. As can be seen from Table 2, the mvalues decreased with increasing concentrations of 4-CPfor both cultures. The percentage inhibition observed onm (the percentage decrease in m value compared to thatof the baseline reactor) for both cultures is shown inFig. 2. The value of IC50 (concentration causing 50%inhibition) on the basis of m was found to be 218mg/Lfor 4-CP-acclimated culture, whereas it was 130mg/Lwhen an unacclimated culture was used. Although aremarkable decrease in the toxic effect of 4-CP wasobserved after acclimation, the m values of the unac-climated culture were higher than those of the accli-mated culture (Table 2). This was also apparent fromthe m values of acclimated and unacclimated culturesreceiving no 4-CP (Table 2). Similar to the observed mvalues, the qðm=Y Þ values of unacclimated culture weremuch higher compared to those of acclimated culture.These results can also be concluded from the timerequired 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) q (mg COD/(mg MLVSSh)) m (1/h)

4-CP-acclimated 0 11 6271 0.7370.15 0.17970.1 0.13170.011

culture 130 25 7478 0.5370.01 0.13770.08 0.07370.007

200 28 6771 0.5770.02 0.11270.07 0.07070.007

300 49 7277 0.4070.08 0.10570.05 0.04270.004

390 76 2471 0.5970.02 0.03370.007 0.02070.003

Unacclimated culture 0 11 6472 0.4770.10 0.780570.1 0.36770.064

57 13 6475 0.5270.08 0.49270.1 0.25670.007

112 22 4671 0.5270.03 0.434670.07 0.22670.004

155 23 3571 0.670.03 0.25670.09 0.15470.008

274 23 4072 0.470.02 0.26570.03 0.10670.002

aTime 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 acclimatedculture due to slower growth and substrate utilizationability. A variety of phenomena have been proposed toexplain the acclimation phase; one is the selection andmultiplication of specialized microorganisms during theacclimation phase (Wiggins et al., 1987; Rittmann andMcCarty, 2001). The results of our study suggest that4-CP-tolerant-microorganims, having slower growthability, were selected. In support of this, Buitron et al.(1998) claimed that acclimated activated sludge wascomposed of some bacteria having low growth-ratecoefficients, but their participation was essential forefficient biodegradation of chlorophenols.

For the acclimated culture, the COD removalefficiencies achieved for the studied range of 4-CP(130–300mg/L) were almost the same and slightlyhigher than that observed for the baseline reactor ofthe acclimated culture (Table 2). Hence, it can be saidthat culture acclimated to 130mg 4-CP/L (whichcorresponds to the IC50 value on the basis of mdepression for unacclimated culture) was not adverselyaffected by 4-CP up to 300mg/L on the basis of CODremoval efficiency. On the other hand, in the case of theunacclimated culture, the baseline COD removal effi-ciency decreased from 64 to 46 and 35% when 4-CPconcentration was increased to 112 and 155mg/L,

respectively (Table 2). When toxicity of 4-CP iscompared to that of 2,4-DCP, it can be said that 2,4-DCP is more toxic than 4-CP, as the presence of 2,4-DCP resulted in lower COD removal efficiency (Sahin-kaya and Dilek, 2002). Similarly, Buitron et al. (1998)reported that degradation of 4-CP by acclimatedactivated sludge is faster compared to that of 2,4-DCP, possibly due to a more toxic effect of 2,4-DCP onmicroorganisms.

As seen from Table 2, the Y value of 0.73mgMLVSS/mg COD for the baseline case of acclimatedculture was surprisingly high. The reason for obtainingsuch a high Y value may be attributed to the fact thatenergy gained from substrate utilization was channeledinto biomass growth rather than maintenance when 4-CP was excluded from the medium. When 4-CP wasadded to the growth medium at different initialconcentrations, the Y values decreased to between 0.40and 0.59mg MLVSS/mg COD for the acclimatedculture. In the case of unacclimated culture, the Y valuein the absence of 4-CP was 0.47mg MLVSS/mg CODand it ranged between 0.4 and 0.6mg MLVSS/mg CODin the presence of 4-CP at different concentrations(Table 2). Therefore, these observations led us toconclude that for both cultures, Y values did notcorrelate to the initial 4-CP concentration. Also, it wasobserved that in the presence of 4-CP the average Y

value was observed to be 0.5270.085 and0.5170.082mg MLVSS/mg COD for acclimated andunacclimated culture, respectively. Therefore, for bothcultures Y values were almost constant in the presenceof 4-CP. Similarly, Saez and Rittmann (1993) reportedthat the Y value for phenol was not affected by thepresence of 4-CP. They attributed the reason for thisobservation to the fact that the biomass was shuntingabout 0.5% of the electrons gained by phenol oxidationto regeneration of NADPH for 4-CP transformation,and this very low diversion of electrons did not have anysignificant effect on Y : In contrast, Hill et al. (1996)found that the total biomass yield of A. eutrophus was


dropped drastically when 4-CP was present in thegrowth medium. They observed that the biomass yielddecreased to 0.36 in the presence of 60 ppm 4-CPcompared to 0.60 when phenol was present as the solesource of carbon. Compared with the 2,4-DCP caseconducted earlier (Sahinkaya and Dilek, 2002), the Y

values decreased with increasing 2,4-DCP for theacclimated culture (Sahinkaya and Dilek, 2002), hence,a certain effect of 2,4-DCP concentration variation onthe biomass yield was obvious, unlike 4-CP.

In regard to the removal of 4-CP, no removal wasdetected within 30 h in reactors inoculated with theunacclimated culture (data were not shown). However,when the acclimated culture was used, nearly completeremoval was observed within 24 and 48 h for 130 and200mg 4-CP/L, respectively (Fig. 3). Hence, resultssuggest that degradation of 4-CP by activated sludgerequires acclimation of biomass. This phenomenon wasalso reported in the studies of Buitron and Gonzalez(1996) and Buitron et al. (1998), for the degradation of4-CP, and Sahinkaya and Dilek (2002) for the degrada-tion of 2,4-DCP. Although 80% removal of 200mg4-CP/L could be achieved within 1 day, no remarkabledegradation of 300mg 4-CP/L was observed within 1day. However, following the first day of incubation,4-CP removal rate increased sharply and almostcomplete removal of 300mg/L was achieved at the endof the second day. This can be attributed to the fact thatthe culture had been acclimated to 130mg 4-CP/L in thefed-batch reactor; therefore, a longer time was requiredfor adaptation to the higher 4-CP concentrations.Furthermore, 4-CP degradation was not observed atall within 76 h when the initial 4-CP concentration was390mg/L. This observation indicates that cultureacclimated to 130mg 4-CP/L could degrade 4-CP upto 300mg/L; hence acclimation to higher 4-CP concen-trations seems to be required in order to remove 4-CPconcentrations higher than 300mg/L.

As can be inferred from Fig. 3, the removal of 4-CPoccurs in two phases, first a slow and then a rapid or

Fig. 3. Variations in 4-CP concentration with time for the acclimated

culture in the presence of peptone.

accelerated removal phase. The specific degradationrates (SDR) of 4-CP at the accelerated phases werecalculated for each initial 4-CP concentration and Fig. 4was prepared to show the relation between them. Thisfigure shows that SDR of 4-CP remained nearlyconstant up to 300mg/L and decreased sharply whenthe 4-CP concentration was increased further to 390mg/L. As concerns the magnitudes, in our study, very highSDRs of 4-CP (102–717mg 4-CP/(gMLVSS day) wereobserved when the acclimated culture was used. Theseresults are in good agreement with those of Buitron et al.(1998), in which the SDR of 4-CP for acclimatedactivated sludge culture was found to be 784mg/(gMLVSSday), which is much higher than the SDRof 1.8mg chlorinated phenols/(gMLVSSday) for anunacclimated activated sludge. Similarly, Sahinkaya andDilek (2002) had observed the removal rates of 226.5and 129mg 2,4-DCP/(gMLVSSday) for the initialconcentrations of 76.6 and 194.5mg/L of 2,4-DCP,respectively. In contrast, Vallecillo et al. (2000) reportedvery low degradation rates of 4-CP (1.2–4.2mg 4-CP/(gMLVSSday) and 2,4-DCP (1.2–1.7mg 2,4-DCP/(gMLVSSday), with no differences in the toxic elimina-tion rate between first and second feed, which could betaken as an indication of ‘‘no effect of acclimation’’ intheir study. Observed SDR values for acclimatedactivated sludge are also much higher compared tothose for pure strains of other studies. Buitron et al.(1998) reported that SDR of 4-CP for Chryseomonas

luteola, Aeromonas sp., Pseudomonas sp., and Flavimo-

nas oryzihabitans were 39, 47, 37, and 44mg/(gMLVSSday) compared to 784mg/(gMLVSS day)for acclimated activated sludge. In the study of Wangand Loh (1999), when the medium pH was kept between6.5 and 7.5, the degradation rates of 4-CP at initialconcentrations of 100 and 200mg/L by Pseudomonas

putida were observed as 9–11 and 10mg/(L h), respec-tively, in the presence of glucose as a growth substrate.On the other hand, when pH of the medium was notregulated, the pH quickly dipped below 4.5, conse-quently stopping the further transformation of 4-CP,and the average transformation rate of 4-CP at an initial

Fig. 4. Specific degradation rate (SDR) of 4-CP for the acclimated

culture at various concentrations of 4-CP.

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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 50mg/L).


concentration of 200mg/L was only about 3mg/(L h). Inour study the pH of the medium was between 7 and 8throughout the study, and the degradation rates of 4-CPwere observed as 8.15, 10.15, 10.22, and 1.03mg/(L h)for initial concentrations of 130, 200, 300, and 390mg/L,respectively. Therefore, our findings are in goodagreement with the literature findings. Based on theabove results, it is apparent that the increase in 4-CPfrom 130 to 300mg/L has no significant effect on theaverage 4-CP transformation rate. In another study, Luet al. (1996) reported that the initial 4-CP removal ratesby Pseudomonas testosterone, Aeromonas radiobacter,

P. putida, and Pseudomonas aeruginosa were 10, 1.2, 1.1,and 2.9mg/(L day) even at 10mg/L. Therefore, thestudied concentrations and degradation rates are verylow compared to those in our study. In order to increase4-CP removal efficiency and rate, Buitron et al. (1998)used a mixed culture formed by selected pure strainshaving the ability to degrade 4-CP and they observedthat the biodegradation capacity of the mixture ofisolates was much lower than for the acclimatedactivated sludge. Therefore, a good biodegradationsystem requires the maintenance of a wide range ofbacterial species at significant concentrations in order toadapt to transient fluctuations in concentrations ortypes of cometabolites that may become present inindustrial effluents (Buitron et al., 1998).

Further comparison of this study with the literaturereports revealed that in contrast to the findings of Wangand Loh (2000), Kim and Hao (1999), and Hill et al.(1996), which pointed to the necessity of phenolsupplementation as the primary substrate for thedegradation of 4-CP, cometabolic degradation of 4-CPis possible using the acclimated culture in the absence ofphenol. Thus, in our study, it was shown that a nontoxicorganic compound, peptone, can be used during thecometabolic degradation of 4-CP and the use of phenol,which is also a toxic compound, could be avoided. Thereare some supportive literature reports on the disadvan-tage of using phenol as the primary substrate (Wang andLoh, 1999; Bali and S-engul, 2002). In the case of usingconventional growth substrate, the cometabolic enzymesrequired for 4-CP transformation were most probablyinduced by 4-CP; cofactor NADH required for 4-CPtransformation could be efficiently formed through theoxidation of conventional growth substrate (Wang andLoh, 1999). Hence, it can be inferred that, in our study,after acclimation, the utilization rate of peptoneincreased and NADH was quickly regenerated, conse-quently facilitating the transformation of 4-CP.

3.2. Treatment of 4-CP when present as the sole organic

carbon source

In addition to the experimental studies discussedabove, degradation of 4-CP in the absence of primary

substrate was also investigated since the addition of aprimary substrate would result in a substantial increasein the overall treatment process cost. Therefore, it isworth devoting attention to the degradation of 4-CPwhen it is present as the sole carbon and energy source.In this set of experiments, in addition to an unaccli-mated and a 4-CP-acclimated culture, a culture pre-viously acclimated to 2,4-DCP was used. In thelatter case, 4-CP was again the only carbon and energysource (i.e., no 2,4-DCP was present). In this set ofexperiments, initial biomass concentration was around50mg/L.

Time-course variations of 4-CP are given in Figs. 5and 6, for the acclimated and the unacclimated culture,respectively. Fig. 5 shows that 50 and 140mg 4-CP/Lcould be removed almost completely within 1 and 3days, respectively, without any lag phase when 4-CP-acclimated culture was used. Although 200mg 4-CP/Lcould also be removed completely within 3 days, theinitial removal rate of 4-CP was lower. On the otherhand, the use of 2,4-DCP-acclimated culture for thedegradation of 138mg 4-CP/L resulted in completeremoval within 5 days. About 3 days of lag period wasrequired for 2,4-DCP-acclimated culture, whereas no lagphase was required when 4-CP-acclimated culture wasused. When the unacclimated culture was used, 4-CPremovals achieved were only 44% and 30% for theinitial 4-CP concentrations of 26 and 130mg/L,respectively, at the end of 18 days (Fig. 6). Therefore,it can be stated that 2,4-DCP-acclimated cultureexhibited less ability than 4-CP-acclimated culture, butbetter ability than the unacclimated culture, toward theremoval of 4-CP. The ability of 4-CP-acclimated cultureto use 2,4-DCP was also shown by Sahinkaya and Dilek(2002). Similarly, Lu et al. (1996) reported that if aspecies can effectively metabolize one type of chlor-ophenols, it is reasonable to expect that this species alsocan effectively utilize other structurally analogouschlorophenols.

ARTICLE IN PRESS

Fig. 6. 4-CP concentrations with time for reactors in which 4-CP

served as the sole organic carbon source for the unacclimated culture.

Fig. 7. Time-course variations in 4-CP and dependence of SDR on

initial 4-CP concentration for acclimated culture (initial cell concen-

tration was 200mg/L).

E. Sahinkaya, F.B. Dilek / Environmental Research 99 (2005) 243–252250

The maximum degradation rates of 4-CP by the 4-CP-acclimated culture in absence of peptone were found tobe 2.56, 1.93, and 2.9mg/(L h) for initial 4-CP concen-trations of 50, 140, and 200mg/L, respectively, com-pared to the maximum degradation rates of8.15–10.22mg/(L h) for initial 4-CP concentration ofbetween 130 and 300mg/L by acclimated culture in thepresence of peptone. The maximum 4-CP degradationrate for 130mg/L by an unacclimated culture in theabsence of peptone was 0.53mg/(L h), which is 4 and 15times lower than that by the acclimated culture in theabsence and the presence of peptone, respectively. Theobserved higher degradation rate in the presence ofpeptone was due to increased biomass concentrationbecause peptone was responsible only for biomassproduction and did not cause any increase in SDR of4-CP, which is discussed later.

3.3. Development of kinetic model

Degradation of 4-CP in the absence of peptone wasalso investigated at higher initial biomass concentrationsto investigate the effect of initial biomass concentrationand obtain kinetic parameters. In this set of experi-ments, initial biomass concentration was kept high toaccelerate degradation rate of 4-CP and avoid furtheracclimation of culture at longer lag-phase durations,which may affect observed biokinetic parameters.Adaptation of cells at prolonged lag phase duringphenol degradation was also reported in the study ofSaez and Rittmann (1993). They reported that acclima-tion of cells to high concentrations of phenol during theassay caused some change in kinetic parameters due to areduced inhibitory effect. In this context, in ourexperimental design, in order to avoid so much changein culture history and correctly estimate kinetic para-meters, degradation experiments were carried out at aninitial biomass concentration of 200710mg/L. There-fore, 4-CP/biomass ratios ranged between 0.05 and 0.94on COD basis.

Fig. 7 shows time-course variation in 4-CP concentra-tion with time and SDR at various initial 4-CP

concentrations. As can be seen from the figure, completeremoval of 4-CP was achieved for all concentrationsstudied. Also, it was observed that the time required tosee complete degradation increased exponentially(r2 ¼ 0:97) with increasing 4-CP concentrations (datanot shown). Compared to Fig. 5, it can be clearlyconcluded that increased initial concentration of bio-mass caused an increase in degradation rate of 4-CP.For example, at an initial concentration of about140mg/L 4-CP, around 3 days was required to achievecomplete degradation when initial biomass concentra-tion was around 50mg/L, whereas around 1 day wasrequired to completely remove 160mg/L 4-CP wheninitial biomass concentration was around 200mg/L.Similarly, the degradation rate of 4-CP increasedfrom 1.93 (for 140mg 4-CP/L) to around 4.5mg/(L h)(for 160mg 4-CP/L) with increasing initial biomassconcentration.

Fig. 7 also shows the relation between specificdegradation rates and initial 4-CP concentrations. Thedecreased 4-CP degradation rate with increasing initial4-CP concentration implies that 4-CP acts as aninhibitor. Therefore, the Haldane substrate inhibitionwas used to model the inhibitory effect of 4-CP on itsown transformation. Application of experimental datato the Haldane equation gave excellent fit to ourexperimental data (Fig. 7) as r2 was observed to be0.986. The least-square error method with the help ofMathlab 6.5 was used to obtain kinetic parameters. The

ARTICLE IN PRESS

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 200mg 4-CP/L.


Haldane parameters for mixed culture acclimated to 4-CP were obtained as qm ¼ 41:17mg 4-CP/(gMLVSS h),K s ¼ 1:104mg=L; and K i ¼ 194:4mg=L: Therefore, theobserved kinetic equation is

dðSÞ

dðtÞ¼

41:17S X

1:104þ S þ S2

194:4

: (1)

Wang and Loh (2001) reported that in 4-CPdegradation, addition of growth substrate, which isstructurally dissimilar to 4-CP, does not affect specificdegradation of 4-CP and it is responsible only for morecell production. In this context, the validity of ourkinetic model for 4-CP degradation was checked byapplying the observed model to one set of experimentsin which peptone (500mg/L COD) was available as theprimary growth substrate. In this set of experiments,initial biomass and 4-CP concentrations were 25 and130mg/L, respectively. Change in biomass concentra-tion was modeled using a logistic growth equation(Shuler and Kargi, 1992). The equation is

dX

dt¼ kX 1�

X

K

� �; (2)

where k and K are growth-associated constant andcarrying capacity of the system, respectively. Theintegration of Eq. (2) yields the logistic curve

X ¼X 0ekt

1� X 0

Kð1� ektÞ

;

where X 0 represents initial biomass concentration.Experimental results gave the carrying capacity as282.6mg/L and k was determined to be 0.252270.03 h�1 (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, time-course variations in 4-CP and biomass can be predicted(Fig. 8). Observed differential equations were numeri-cally solved using POLYMATH 4.02. The program usedthe Runge–Kutta–Fehlberg numerical integration rou-tine. Change in biomass measured by OD was convertedto mg/L MLVSS using the linear relation. It can be seenfrom the figure that time-course variations in bothbiomass and 4-CP were reasonably predicted. Therefore,similar to results of Wang and Loh (2001), the presenceof peptone did not increase the specific degradation rateof 4-CP and it was responsible only for the productionof biomass.

During the batch degradation studies, a yellowishcolor accumulated during the degradation of 4-CP dueto production of CHMS, indicating meta cleavage of4-CP (Saez and Rittmann, 1991; Wang and Loh, 1999;Farrell and Quilty, 1999). The color of the filteredmedium was measured at 380 nm and it was observedthat the intermediate concentration reached its max-imum value when 4-CP was just about completely

removed (data not shown). Also, the maximum absor-bance value showed an increasing trend with increasinginitial 4-CP concentration. After observation of the peakvalue, a sharp decrease in absorbance to its originalvalue indicated complete degradation of 4-CP via themeta-cleavage pathway. HPLC results of influent andeffluent samples also indicated complete removal of4-CP without any by-product observation. As anexample, Fig. 9 shows HPLC diagrams of one selectedbatch experiment.

The results of this study indicated that the toxiceffects of 4-CP decreased remarkably on the basis of mand COD removal efficiency, after acclimation. It wasshown that in the cometabolic degradation of 4-CP, anontoxic organic compound, peptone, can be usedinstead of phenol. Although 4-CP removal by anunacclimated culture was negligible, efficient removalvia the meta-cleavage pathway by the acclimated cultureboth in the presence and in the absence of peptone wasobserved. The Haldane equation seems to be anadequate expression as 4-CP inhibits its own transfor-mation at high concentrations. It was observed thatpeptone did not affect the specific degradation rate of4-CP and it was responsible only for more cellproduction. Also, 4-CP could be degraded by 2,4-DCP-acclimated culture, although not as effectively asby 4-CP-acclimated culture.


Acknowledgments

The authors express their gratitude to the Middle EastTechnical University Research Fund, which supportedthis work (Project AFP2000-03-11-03).

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