Biochemical Engineering Journal 24 (2005) 243–247

The biodegradation of phenol at high initialconcentration by the yeastCandida tropicalis

Jiang Yana,b, Wen Jianpinga,∗, Li Hongmeia,Yang Sulianga, Hu Zongdinga

a Department of Biochemical Engineering, School of Chemical Engineering and Technology,Tianjin University, Tianjin 300072, PR China

b School of Life Sciences and Chemistry, Harbin college, Harbin 150016, PR China

Received 5 February 2003; accepted 5 February 2005

Abstract

StrainCandida tropicaliswas isolated from acclimated activated sludge, and was identified as a member of the genusCandida. Phenolbiodegradation using a pure culture ofC. tropicaliswas studied. The results showed thatC. tropicalishas pretty high phenol degradationp nedp the stepi d by cell forg tics oft l.T havior of thep©

K

1

etrpitcmgt

i

or-sole

didus-

t high-

arac-g

wthed byntra-

1d

otential, which could thoroughly degrade the phenol of 2000 mg l−1 in the mineral salt medium within 66 h. High inoculum volume lessehenol toxic property for the cells and increased phenol biodegradation velocity. However, for a certain starting inoculum, with

ncrease of phenol concentration, substrate inhibition was obviously enhanced, and more phenol consumption was not assimilaterowth, but was used to counteract strong substrate inhibition. In addition, the cell growth and phenol degradation intrinsic kineC.ropicalis in batch cultures were also investigated over a wide range of initial phenol concentrations (0–2000 mg l−1) using Haldane modehe results received in these experiments demonstrated that the Haldane kinetic model adequately described the dynamic behenol biodegradation by the strain ofC. tropicalis.2005 Elsevier B.V. All rights reserved.

eywords: Candida tropicalis; Haldane’s equation; High phenol concentration; Inoculum volume; Phenol degradation; Substrate inhibition

. Introduction

Phenol at high concentration is widely distributed asnvironmental pollutants due to its common presence in

he effluents of many industrial processes, including oilefineries, ceramic plants, coal conversion processes andhenolic resin industries[1–4]. Once wastewater contain-

ng phenol is discharged into the receiving body of wa-er, it would endanger fish life, even at relatively lowoncentration, e.g. 5–25 mg l−1 [5,6]. Therefore, the re-oval of phenol from industrial aqueous effluents is ofreat practical significance for the environmental protec-

ion.By contrast with physical and chemical methods, biolog-

cal methods of phenol removal are preferable in wastewater

∗ Corresponding author. Tel.: +86 22 27890492; fax: +86 22 27890492.E-mail address:jpwen@tju.edu.cn (W. Jianping).

treatment process as the relatively low processing costs[7–9].In spite of phenolic toxic properties, a number of microganisms can utilize phenol under aerobic condition assources of carbon and energy. Hofmann and Krueger[10],Vallini et al.[11], Alexievaa et al.[12] and Santos and Linar[13] have isolated and characterized some fungi from intrial effluents for mineralizing 100–1000 mg l−1 phenol. Fi-alova et al.[14] reported the yeastCandida maltosafor itsphenol-degrading potential up to 1700 mg l−1. However, lit-tle has been known about the biodegradation of phenol ainitial concentration larger than 1700 mg l−1 by microorganisms.

Objectives of the present study are to isolate and chterize the yeastC. tropicaliswith potential for biodegradinphenol at initial concentration larger than 1700 mg l−1, andto study the intrinsic kinetics of the microorganism groand phenol degradation, which is defined as not influenctransport processes and only concerned with the conce

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2005.02.016

244 J. Yan et al. / Biochemical Engineering Journal 24 (2005) 243–247

Nomenclature

A growth associated constant for phenol con-sumption

B non-growth associated constant for phenolconsumption (h−1)

CS phenol concentration (mg l−1)CX cell concentration (mg l−1)Ki inhibition constant for cell growth (mg l−1)Ks saturation constant for cell growth (mg l−1)m maintenance energy coefficient (h−1)t time (h)YP/S product yield coefficientYX/S biomass yield coefficient

Greek symbolsα growth associated constant for product forma-

tionβ non-growth associated constant for product

formationγ rate of reaction (mg l−1 h−1)γP product formation rateγX cell growth rateµmax maximum specific growth rate (h−1)µX specific growth rate (h−1)

tion of reactants, temperature and the character of the solvent,using Haldane’s equation[15], respectively.

2. Materials and methods

2.1. Acclimation of activated sludge

Activated sludge was collected from a municipal gasworksin China during the month of September 2003 and enrichedfor a period of 10 weeks using phenol as the sole carbonsource in the mineral medium.

2.2. Microbial strain, culture media and cultivationconditions

Diluted activated sludge was inoculated into shakingflasks with the YEPD medium[16], and initial pH value wasadjusted to 6.0. After three multiplication cultures, the latecultures were diluted and plated onto agar plates which wereincubated afterwards at 30◦C about 24 h. Phenol degradationwas tested to compare phenol-degrading potential of theseindividual isolates after being inoculated on agar slants. Adominant colony type was purified through several transfersto the plates again.

umw ).

Liquid mineral medium contained: 0.4 g l−1 K2HPO4,0.2 g l−1 KH2PO4, 0.1 g l−1 NaCl, 0.1 g l−1 MgSO4,0.01 g l−1 MnSO4·H2O, 0.01 g l−1 Fe2(SO4)3·H2O,0.01 g l−1 Na2MoO4·2H2O, 0.4 g l−1 (NH4)2·SO4, pH 6.0.Phenol was filter-sterilized through membranes (pore size of0.2�m) and added to the medium before inoculation[5].

The strain was identified based on physiological and bio-chemical tests and Biolog Microbial Identification System[17].

2.3. Phenol degradation

The startup of the experiments was obtained by inoculat-ing 10 ml YEPD medium (phenol concentration: 100 mg l−1)with microbial strain from nutrient agar slants. After 16 hof incubation, 2 ml this cell culture was added to 100 mlfresh YEPD medium. Cells grown in late phase of thegrowth curve were harvested as inoculum. To investigatethe influence of inoculum volume on phenol degradation,50 ml mineral medium with 1000 mg l−1 phenol were in-oculated with the subculture of 2.5, 5, 7.5 ml, respectively.Besides, for the determination of intrinsic kinetics parame-ters, 2.5 ml subculture was inoculated into 50 ml the min-eral medium containing the varying phenol concentrationfrom 0 to 100 mg l−1 at 20 mg l−1 interval, and from 200 to2 ep kenf

2

bymb suredb thefim iatelya ndedc cellf ncen-t LC)u 18c sp e of1 tor(

3

3

ed,a asedo the

Liquid cultures were grown in the minimal salt mediith 500 mg l−1 phenol at 30◦C on a rotary shaker (200 rpm

000 mg l−1 at 200 mg l−1 interval, respectively. During theriod of batch culture, all samples were periodically ta

or biomass and phenol concentration.

.4. Analytical methods

Cell density was monitored spectrophotometricallyeasuring the absorbance at wavelength 600 nm[18]. Theniomass concentrations on a dry weight basis were meay filtering the cell suspension with the filler and dryinglter paper and cells to a constant weight for 24 h at 105◦C. Toeasure concentration of phenol undegraded, immedfter measurements of optical density, samples of suspeulture were centrifuged at 7500 rpm for 10 min. Theree supernatants were used to determine the phenol coration by high performance liquid chromatography (HPsing a LabAlliance (model SeriesIII) system, with a Column (250 mm× 4.6 mm, LabAlliance, USA). Elution waerformed with 400/300 (v/v) methanol/water at a flow rat.0 ml min−1, and detection was realized with a UV detecModel 500, LabAlliance, USA) at 280 nm.

. Results and discussion

.1. Isolation and identification of microbial species

A total of 68 fungal isolates from plates were obtainmong which 22 were selected for further screening bn phenol tolerance. Of the 22 isolates, one strain with

J. Yan et al. / Biochemical Engineering Journal 24 (2005) 243–247 245

Fig. 1. The effect of inoculum volume on phenol degradation and the cellgrowth.

maximum degradation velocity was chosen to use for follow-ing experiments.

The strain was identified by Institute of Microbiology, Chi-nese Academy of Sciences. Identification studies on the strainrevealed clearly the presence ofC. tropicalis.

3.2. Phenol degradation

Fig. 1 indicated the effect of inoculum volume on phenoldegradation. The cells inoculated with 15% starting inocu-lum underwent a short lag phase, and manifested the highphenol-degrading velocity. A 1000 mg l−1 of phenol was en-tirely degraded within 32 h, with the time consumed muchshorter than that of the other two (33.5 and 34.5 h, respec-tively), however, with the more biomass yielded. The rea-sons were accounted as following: apart from the differentinitial cell concentrations, more phenol consumed and morenutrient in YEPD medium was utilized to synthesize newcells.

Ft2

Fig. 2 showed cell growth and phenol degradation atthe phenol of 1600–2000 mg l−1 with 5% starting inocu-lum. It can be seen that 1600 and 1800 mg l−1 phenol couldbe completely degradated byC. tropicalis within 54 and59 h, respectively. Compared with the time consumed inthe sample of 1800 mg l−1, more than 7 h was spent inthe sample 2000 mg l−1 for that matter. Furthermore, thebiomass yields were not in accordance with the step in-crease of phenol concentration, as from 1600 to 1800 mg l−1

biomass yields increased 37.44 mg l−1 while from1800 to2000 mg l−1 biomass yields merely increased 26.97 mg l−1.It exhibited a remarkable augment of substrate inhibition,which could be also demonstrated by the longer lag phase ofcell growth. Hence, cell growth was out of proportion to phe-nol degradation as former report, especially at high phenolconcentration[19], although phenol was consumed mainlyfor assimilation into biomass and energy for cell growth andmaintenance[20]. Besides, the production and accumulationof various intermediates may also be responsible for the de-creased cell mass yield[21,22].

3.3. Intrinsic kinetic study

Batch cultures ofC. tropicaliswere conducted in the min-eral medium containing initial phenol concentrations rangingf −1 alp lateda

µ

wr

tiono d forap

µ

wua st-s asedo enolb f therw entald

wthr con-cI thr theri lues

ig. 2. The cell growth and phenol degradation byCandida tropicalisinhe mineral medium containing initial phenol from 1600 to 2000 mg l−1 at00 mg l−1 interval with 5% starting inoculum.

rom 0 to 2000 mg l . For each flask with a certain initihenol concentration, the specific growth rate was calcus

X = γX

CX= dCX

dt

1

CX(1)

hereµX is the specific growth rate (h−1),γX the cell growthate, andCX is the cell concentration (mg l−1).

Because of the inhibition of high phenol concentran the cell growth, the Haldane’s equation was selectessessing the dynamic behavior ofC. tropicalis grown onhenol:

X = µX,maxCS

Ks + CS + (C2S/Ki )

(2)

hereCS is the phenol concentration (mg l−1). The val-es of the parametersµX,max= 0.48 h−1, Ks = 11.7 mg l−1

ndKi = 207.9 mg l−1 were derived using a nonlinear leaquares regression analysis of the software of Matlab bn the experimental data obtained in the tests of phiodegradation. The value of the squared two-norm oesidual at these parameters was a small value (1.21× 10−3),hich indicated the regression curve agreed the experimata very well.

The comparison between calculated specific cell groates and experimental data at different initial phenolentration varied from 0 to 2000 mg l−1 was shown inFig. 3.t can be seen fromFig. 3that the maximum specific growate occurred at low phenol concentration. And with furncrease of initial phenol concentration, much lower va

246 J. Yan et al. / Biochemical Engineering Journal 24 (2005) 243–247

Fig. 3. Specific growth rate as a function of the different initial phenol con-centration.

of the specific growth rates were obtained. The phenomenonwas solely due to intense substrate inhibition at high phenolconcentration.

Analyzing the utilization of the substrate in cells in moredetail, the consumption of substrate for growth and for main-tenance and also, for product formation if possible has to beconsidered[23]. The substrate consumption rate of phenolbiodegradation is

γS = 1

YX/SγX + mCX + 1

YP/SγP (4)

whereγP =αγX +βCX is the product formation rate and be-causeYX/S, m, YP/S, α, β are all constants, Eq.(4) can bereduced to

γS = AγX + BCX (5)

F mineds

or

µS = AµX + B (6)

A andB are all kinetic constants and they were regressedusing Matlab based on the experimental data,A= 0.823,B= 0.277 h−1 (R2 = 0.981).

Fig. 4 was the comparison between the prediction of thedegradation kinetics and the experimentally determined spe-cific degradation rates ofC. tropicalisat different initial phe-nol concentrations from 0 to 2000 mg l−1, from which it couldbe seen that the simulated values of degradation kineticsagreed well with the experimental data.

4. Conclusions

The strain ofC. tropicaliswas isolated from acclimatedactivated sludge, and identified as a member of the genusCandida. StrainC. tropicaliscould degrade the phenol con-centration beyond 1700 mg l−1.

The intrinsic kinetics of phenol biodegradation byC.tropicalis was investigated at the initial phenol concentra-tion varied from 0 to 2000 mg l−1, the temperature of 30◦C,and the initial pH of 6.0. The intrinsic kinetic models forthe specific growth rate and the specific degradation ratewa

A

portp ofC

R

ctionsng.

ation–23.ations

ndphere

he-4 (4)

d in-

07.cti-1999)

ig. 4. Comparison between kinetic prediction and experimental deter

pecific degradation rates at different initial phenol concentrations.

ere proposed asµX = 0.48CS/(11.7 + CS + C2S/207.9)

ndµS = 0.823µX + 0.277, respectively.

cknowledgements

The authors wish to acknowledge the financial suprovided by the National Natural Science Foundationhina (No. 20336030).

eferences

[1] K. Bandhyopadhyay, D. Das, P. Bhattacharyya, B.R. Maiti, Reaengineering studies on biodegradation of phenol byPseudomonaputidaMTCC 1194 immobilized on calcium alginate, Biochem. EJ. 8 (2001) 179–186.

[2] I. Alemzadeh, F. Vossoughi, M. Houshmandi, Phenol biodegradby rotating biological contactor, Biochem. Eng. J. 11 (2002) 19

[3] Z. Aleksieva, D. Ivanova, T. Godjevargova, B. Atanasov, Degradof some phenol derivatives byTrichosporon cutaneumR 57, ProcesBiochem. 37 (2002) 1215–1219.

[4] V. Kavitha, K. Palanivelu, The role of ferrous ion in fenton aphoto-fenton processes for the degradation of phenol, Chemos55 (2004) 1235–1243.

[5] M. Kibret, W. Somitsch, K.H. Robra, Characterization of a pnol degrading mixed population by enzyme assay, Water Res.(2000) 1127–1134.

[6] T.P. Chung, H.Y. Tseng, R.S. Juang, Mass transfer effect antermediate detection for phenol degradation in immobilizedPseu-domonas putidasystems, Process Biochem. 38 (2003) 1497–15

[7] F. Bux, B. Akkinson, K. Kasan, Zinc biosorption by waste avated and digested sludges, Water Sci. Technol. 39 (10–11) (127–130.

J. Yan et al. / Biochemical Engineering Journal 24 (2005) 243–247 247

[8] A. Zumriye, A. Derya, R. Elif, K. Burcin, Simultaneous biosorp-tion of phenol and nickel from binary mixtures onto driedaerobic activated sludge, Process Biochem. 35 (1999) 301–308.

[9] C.C. Wang, C.M. Lee, C.J. Lu, M.S. Chuang, C.Z. Huang, Biodegra-dation of 2,4,6-trichlorophenol in the presence of primary sub-strate by immobilized pure culture bacteria, Chemosphere 41 (2000)1873–1879.

[10] K.H. Hofmann, A.-K. Krueger, Induction and inactivation of phe-nol hydroxylase and catechol oxygenase inCandida maltosaL4 independence on the carbon source, J. Basic Microbiol. 25 (1985)373–379.

[11] G. Vallini, S. Frassinetti, F.D. Andreac, G. Catelani, M. Agnolucci,Biodegradation of 4-(1-nonyl)phenol by axenic cultures of the yeastCandida aquaetextoris: identification of microbial breakdown prod-ucts and proposal of a possible metabolic pathway, Int. Biodeter.Biodegr. 47 (2001) 133–140.

[12] Z. Alexievaa, M. Gerginova, P. Zlateva, N. Peneva, Comparison ofgrowth kinetics and phenol metabolizing enzymes ofTrichosporoncutaneumR57 and mutants with modified degradation abilities, En-zyme Microb. Technol. 34 (2004) 242–247.

[13] V.L. Santos, V.R. Linardi, Biodegradation of phenol by a filamentousfungi isolated from industrial effluents-identification and degradationpotential, Process Biochem. 39 (2004) 1001–1006.

[14] A. Fialova, E. Boschke, T. Bley, Rapid monitoring of the biodegrada-tion of phenol-like compounds by the yeastCandida maltosausingBOD measurements, Int. Biodeter. Biodegr. 54 (2004) 69–76.

[15] J.F. Andrews, A mathematical model for continuous culture of mi-croorganisms utilizing inhibitory substrates, Biotechnol. Bioeng. 10(1968) 707–723.

[16] T.G. Haydee, Ploidy study in Sporothrix schenkii, Fungal Genet.Biol. 27 (1999) 49–54.

[17] E. Heinaru, J. Truu, U. Stottmeister, A. Heinaru, Three types ofphenol andp-cresol catabolism in phenol- andp-cresol-degradingbacteria isolated from river water continuously polluted with pheno-lic compounds, FEMS Microb. Ecol. 31 (2000) 195–205.

[18] F. Zeng, K.Y. Cui, J.M. Fu, G.Y. Sheng, H.F. Yang, Biodegradabil-ity of di (2-ethylhexyl) phthalate byPseudomonas fluorescensFS1,Water Air Soil Pollut. 140 (2002) 297–305.

[19] M. Claußen, S. Schmidt, Biodegradation of phenol andp-cresol bythe hyphomyceteScedosporium apiospermum, Res. Microbiol. 149(1998) 399–406.

[20] E.S. Venkataramani, R.C. Ahlert, Role of Cometabolism in biologicaloxidation of synthetic compounds, Biotechnol. Bioeng. 27 (1985)1306–1311.

[21] P.J. Allsop, Y. Chisti, M.M. Young, G.R. Sullivan, Dynamics ofphenol degradation byPseudomonas putida, Biotechnol. Bioeng. 41(1993) 572–580.

[22] O.J. Hao, M.H. Kim, E.A. Seagren, H. Kim, Kinetics of phenol andchlorophenol utilization byAcinetobacterspecies, Chemosphere 46(2002) 797–807.

[23] H. Feitkenhauer, S. Schnicke, R. Muller, H. Markl, Kinetic param-eters of continuous cultures ofBacillus thermoleovoranssp. A2 de-grading phenol at 65◦C, J. Biotechnol. 103 (2003) 129–135.