Biochemical Engineering Journal 30 (2006) 174183

Biodegradation of high phenol concentratioimmersed membrane bi

. M1, Unvenc

ary 2

Abstract

The effec studdifferent ph shoremoval of p for thconcentratio axim(Ks = 29.54 m h theagreement w rent 2006 Else

Keywords: A embr

1. Introdu

Phenols, i.e. hydroxy compounds of aromatic hydrocarbons,and its derivatives are widely used as raw materials in manypetrochemical industries and petroleum refineries (washing andconditioninpharmaceuers industrirefining, taThus, the pindustrial psents a signdevelopmetrial wastew

As the tocentration (even reducter treatmethe biologimakes the p

Abbreviat Correspon

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thephenolic compounds like granular or biological activated car-bon, H2O2/UV processes, O3/UV processes, Fenton processes(Fe2+/H2O2) solvent extraction, membrane processes. Conven-tional processes have been mostly physicochemical processes

1369-703X/$doi:10.1016/jg of the alkaline or acid products), chemical andtical industries (dyes, pesticides, drugs, . . .) and oth-es like pulp and paper mills, coking operations, coalnnery and foundries (washing of the gas effluents).resence of phenols in water generally comes from thisollution. The increasing presence of phenols repre-ificant environmental toxicity hazard; therefore, the

nt of methods for the removal of phenols from indus-ater has generated significant interest.xicity of phenolic compounds is important, their con-up to several grams per liter) unfortunately inhibits ores microorganisms in municipal biological wastewa-nt plant [1]. The presence of phenols strongly reducescal biodegradation of the other components; whatrocess of degradation of phenols so difficult. Accord-

ions: MBR, membrane bioreactor; RCF, relative centrifugal forceding author. Tel.: +33 442 908 511; fax: +33 491 289 407.dress: benoit.marrot@univ-cezanne.fr (B. Marrot).

but since they cause secondary problems in the effluents (forexample, phenol becomes chlorophenols if chlorination is used),biological treatments are preferred for large-scale removal ofthis type of pollutants. It is one of the reasons why activatedsludge reactors have been widely used for phenol removal fromindustrial wastewater.

2. Biological treatment

2.1. Acclimated activated sludge

Biological treatment is a practical and not very expensivesolution to treat this kind of effluents compared to chemicalone (not need to add chemicals); because various populationsof microorganisms in the activated sludge are able to degradeorganic compounds and most of effluents can be biologicaldegraded. The species most often present are pseudomonas,avobacterium, achromobacter, rhomobacterium, azobacter,micrococcus, bacillus alkaligenes, arthrobacter, ycobacterium,aeromonas, nocardia and lophomonas. Their respective propor-

see front matter 2006 Elsevier B.V. All rights reserved..bej.2006.03.006B. Marrot , A. Barrios-Martinez, PLaboratoire de Procedes Propres et Environnement UMR-CNRS 618

Europole de lArbois, BP 80, 13545 Aix en ProReceived 29 June 2005; received in revised form 17 Janu

t of adaptation of mixed culture in the phenol biodegradation has beenenol concentrations from 0.5 to 3 g L1. Biological treatment has beenhenol. High concentrations of phenol are inhibitory for growth; so it isns. Haldane kinetics model for single substrate was used to obtain mg L1) and substrate inhibition constant (Ki = 72.45 mg L1). Althoug

ith those reported in the literature for phenol removal abilities in diffevier B.V. All rights reserved.

ctivated sludge; Phenol; Biodegradation; Kinetic model; Haldane; Immersed m

ction ing ton by activated sludge in anoreactoroulin, N. Roche

iversite Paul Cezanne dAix-Marseille III,e Cedex 04, France006; accepted 28 March 2006

ied. The degradation experiments have been conducted atwn to be economical, practical and it leads to a completee rates of substrates utilization that are greater at low initialum specific growth rates (m = 0.438 h1), half saturationconcentration in phenol is significant, these results are in

systems and the Haldane model is still acceptable.

ane bioreactor

literature, several processes are used to remove

B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183 175

Nomenclature

kdKiKsMLSSStX

Greek le

m

Subscrip0

tion will deand the potivated, theenergy.

Neverthpounds is nthe microor200 mg Lof the micare relatedand, of couDuring thiscialized mithe microortypes of Grthe acclimaFlavomonaother microsp., Sarcintobacter anT. cutaneumdegrading mmainly speconsiderabPseudomonthe higheststudies upobeen donemated activefficientlynitude fastliquors hadvated sludg

To obtaimicroorganthe cells afor the fluiculture (freuous proce

batch reactor is finished. In comparison of these two processes[9,11,13,14] it seems that better phenol degradation efficiency

inedtankforgradecombe oilize

f freemedrmedation

ffect

nol dactor

temhighe anonsa decer e

n phherver,

freeet a

ocesells aylicof btrati

n betorsht rencreThe

nol oxygedecay coefficient (h1)substrate inhibition coefficient (mg L1)half saturation coefficient (mg L1)mixed liquor suspended concentration (g L1)concentration of substrate (mg L1)time (h)concentration of biomass (mg L1)

ttersspecific growth rate (h1)maximum specific growth rate (h1)

tinitial value

pend mainly on the specific substrate concentrationtentiality of growth. When microorganisms are cul-y consume substrates for their growth and for their

eless, biological treatment of the phenolic com-ot easy because of the proper toxicity of phenol forganisms. Even with weak concentrations (lower than

1) the phenolic compounds can cause the inhibitionrobial growth. The limits of the biological processto the acclimation of the biomass to degrade phenolrse, to the variability of the wastewater composition.phase there is a selection and a multiplication of spe-

croorganisms. Buitron et al. [2] isolated and identifiedganisms responsible for the phenol degradation. Fouram-negative unicellular bacteria were obtained fromted consortium: Aeromonas sp., Pseudomonas sp.,s oryzihabitans andChryseomonas luteola. There areorganisms able to degrade phenol like: Alacaligenesas, Desulfovibrio sp., Bacillus alkaligenes, Acine-d more [3]. Alexievaa et al. [4] demonstrated that

R57 has all the properties of an efficient phenol-icroorganism. Two groups of degrading bacteria are

cified and used: the Rhodococci (Rhodococci shows

is obtastirredeffect;can delevel bcouldimmobcase o

with aof intedegrad

2.2. E

Phement fing thefind athe ratdeviaticause

the lowbetweethe higMoreoter forChungtwo prlized ccarboxthe pHconcen

pH cathe facA sligation i[5,10].of phecient ole morphological) like Rhodococcus spp. [58] andads like Pseudomonas putida. P. putida seem to havedegradative potential. That is why a great number ofn the degradation of phenols by these bacteria has

[914]. But opinions are divided, it seems that accli-ated sludge degrades the phenolic compounds morethan the pure strains by one to two orders of mag-er and Annadurai et al. [15] showed that the mixeda best ability for phenol degradation than pure acti-e and P. putida.n a specified biomass from activated sludge, all theseisms are generally used in two different ways, eitherre immobilized within calcium alginate gel beadsdized-bed bioreactor or cells grow as a suspendede cells) for the bioreactor. In a lot of case, the contin-ss is considered when the acclimation step in stirred

significantlnol degradresist pH cposition radecrease inrated as thAksu and Gof the activis predomicould be exion above aon cells besludge so tvated sludg

In conclrange [6.5;with a fluidized-bed bioreactor rather than with a. It shall be explained by the substrate inhibition

example in the study of Chung et al. [14], free cellse phenol only up to about 600 mg L1 whereas thises up to 1000 mg L1 for immobilized cells. But it

bserved the formation of intermediate catechol ford cells; phenomenon has not been detected in thecells. So, it seems that the use of immobilized cells

ium diffusion resistance could be a mean for detectioniates during substrate degradation. Moreover, phenolreaction is slower in the immobilized systems.

of temperature and pH on phenol degradation

egradation seems to be determined by some environ-s such as temperature and pH [9,10,1215]. Regard-perature effect, authors are almost unanimous ander phenol removal efficiency near 30 C. Howeverd the extent of degradation is relatively sensitive tooutside the optimal range [9]. A variation of 5 C mayrease in phenol degradation rate of at least 50% at

nd and almost 100% at the higher end. The differenceenol removal efficiency at 30 C is probably due toproduction of metabolites at this temperature [12].at this temperature, the degradation rate seems bet-than immobilized cell system (1.45 times higher).

l. [14] found an optimal temperature of 30 C for theses but different optimal pH values: 6.8 for immobi-nd 8.0 for free cells. This difference is related to theparts of alginate that attract H+ around them. Hence,uffer solution has to be lowered to provide optimalon of H+ for P. Putida. The follow-up of the mediuman indicator of the phenol degradation and one ofsignificant in the success of the biological treatment.duction is observed as biomass grows and pH vari-ases when the initial phenol concentration increasesdecrease in pH suggests that biological degradationccurs and with a stable pH of about 7 (and a suffi-n supply) phenol was successfully degraded. The pHy affects the biochemical reactions required for phe-ation; tests with pure P. putida could not efficientlyhange [15]. pH medium affects the substrate decom-te and phenol decomposition leads to a considerable

pH. Consequently, phenol degradation is deterio-e medium pH deviates from neutral condition. Foronen [16] pH affects the surface charge of the cells

ated sludge biomass. The surface charge of biomassnantly negative over the pH range of 310. Phenolpected to become negatively charged in phenoxidepH of 9. Below a pH of 3, the overall surface charge

comes positive due to isoelectric point of activatedhe electrostatic attraction between phenol and acti-e biomass will be insignificant [16].usion, it seems that these studies indicate a best pH7.5] for the phenol degradation from effluents.

176 B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183

Table 1Composition of growth mediumAcclimation Biodegradation

of phenolReference

Culture Process T (C) pH Nutrient medium [Phenol](mg L1)

Phenol loadingrate (g L1 d1)

Growth medium(mg L1)

Process

Pseudomonasputida ATCC17484 Biotype B

Batch 30 6.6 Beef extract, 1 g L1;yeast extract, 2 g L1;peptone, 5 g L1; agar,15 g L1; NaCl,5 g L1; K2HPO4,2.39 g L1

51000 0.54 KH2PO4, 420;K2HPO4, 375;(NH4)2SO4, 244;NaCl, 30; CaCl2;MgSO47H2O, 61.4;FeCl24H2O, 4.7

Batch reactorfluidized-bedreactor

[11]

Pseudomonasputida

Batch Glucose, 2 g L1 500 CaCl24H2O, 69.9;NaCl, 8; KNO3,103; NaNO3, 698;MgSO47H2O, 100;NTA, 100;FeSO47H2O, 2;ZnSO47H2O, 0.1;MnSO45H2O;0.043; H3BO3, 0.3;CoSO47H2O, 0.24;CuSO45H2O, 0.01;NiSO47H2O, 0.02;NaMoO42H2O,0.03; Ca(OH)2,0.5;EDTA, 5; KH2PO4,544.4; Na2HPO4,2148.9; (NH4)2SO4,30

Trickling bedreactor

[12]

Pseudomonasputida DSM 548

Batch 26 6.8 Agar 1100 KH2PO4, 420;K2HPO4, 375;(NH4)2SO4, 244;NaCl, 15;CaCl22H2O, 15;MgSO47H2O, 50;FeCl36H2O, 5.4

Batch reactor [10]

Pseudomonasputida

25 6.6 Agar, 11 g L1 500 KH2PO4, 840;K2HPO4, 750;(NH4)2SO4, 488;NaCl, 60; CaCl2,60; MgSO4, 60;FeCl3, 60

Immobilizedbeads

[9])

PseudomonasputidaCCRC14365

Batch 30 7 Beef extract, 3 g L1;peptone, 5 g L1;mineral salt

100 KH2PO4, 420;K2HPO4, 375;(NH4)2SO4, 244;NaCl, 15;CaCl22H2O, 15;MgSO47H2O, 50;FeCl36H2O, 54

Free suspensionImmobilizedCa-alginatebeads

[14]

Pseudomonasputida

Batch 30 7 Beef extract, 1 g L1;yeast extract, 2 g L1;peptone, 5 g L1;NaCl, 5 g L1; agar,15 g L1

Immobilized oncalcium alginate

[17]

Pseudomonasputida ATCC31800 andactivated sludge

Batch 3036 7 to 9 Beef extract, 1 g L1;yeast extract, 2 g L1;peptone, 5 g L1;NaCl, 1 g L1; agar,20 g L1; glucose, 0.5,0.6, 0.7, 0.8 g L1

0.25 (NH4)2SO4,500800 andmedium mineral.**pH 6.25 thePseudomonas putidacould not resist pHchange t = 48 h**phenol loading of0.25 g L1 day

Batch reactor [15]

Activated sludge(2500 mg L1)

Batch 25 300 (NH4)2SO4, 240;K2HPO4, 45;NaOH, 120;MgCl26H2O, 15;CaCl2, 4;FeCl36H2O, 0.6

Activated sludge [18]

B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183 177

Table 1 (Continued )Acclimation Biodegradation

of phenolReference

Culture Pra

Aeromonas sp.Pseudomonsp.; FlavomoryzihabitanChryseomonluteola andactivated slu

0

Pseudomonasputida

0

Trichosporoncutaneum R

RohodococcusDCB-p0610

Activated sludg

From thtions (medparticular sfew reportsbeen done;from a was

The aimto phenol uless favorabof the substTo investiginitial conckinetics usof phenol fimmersed mProcess T (C) pH Nutrient medium [Phenol](mg L1)

;asonass;as

dge

Batch 15 N, P and trace ofelements

40 Phenol 2,4DCP; 2,4,6 TCP

Immobilizedbeads

30 6.6 Agar, 11 g L1 2.610.657Inmovilizedcells

28 6 Beer agar and 0.67%yeast nitrogen bases

0.5 Isopropyl-benzene

0

sp. Inmovilizedon GAC;aginatebeads byentrapment

6 to 8; 4to 7

NaHPO4, 10 g;KH2PO4, 1 g;(NH4)2SO4, 3 g;FeCl3, 0.018; NaCl,0.2 g; CaCl2, 0.08;MgSO47H2O, 0.2 g;yeast extract,0.5 g L1; phenol, 10 g

2000 2fr2frb

e Batch 30 7.2 Phenol variable(7504500)

14

e literature (Table 1) we know that optimum condi-ium, temperature, pH) to acclimatize bacteria to thisubstrate which is the phenol. It is also noticed thaton phenol biodegradation using real effluents hasthat is why we have worked with activated sludge

tewater treatment plant.of this study was double: (i) to acclimatize bacteria

nder experimental conditions easier to implement butle for the growth of these microorganisms (limitationrate, pH and temperature different of the ideality). (ii)ate the possibility of phenol biodegradation at highentrations and to study the microorganism growth

ing AndrewsHaldane model during biodegradationor single substrate. This study is carried out with an

embrane bioreactor.

3. Materia

3.1. Appar

The matium formebetween alamong othflocculationbe less effiis the reasocarried outuninterrupttration holbioreactorhenol loadingte (g L1 d1)

Growth medium(mg L1)

Process

.03 30 of P; 30 of 2,4DCP; 30 of 2,4,6TCP; nutrients

Batch reactor [2]

.250.5 (NH4) NO3, 1;(NH4)2SO4, 0.5;NaCl, 0.5;MgSO47H2O 0.5;KH2PO4, 1.5;K2HPO4, 0.5;CaCl2, 0.01;FeSO47H2O, 0.01in 1 L of solution

Batch reactor [19].250.5 Immobilizedcells

[20]

.9 g L1 dayom GAC.1 g L1 dayom aginateeads

NaHPO4, 10 g;KH2PO4, 1 g;(NH4)2SO4, 3 g;FeCl3, 0.018; NaCl,0.2 g; CaCl2, 0.08;MgSO47H2O,0.2 g; yeast extract,0.5 g L1; phenol,10 g

Continuousreactor

[5]

.44, 2.40 and

.32MgSO47H2O, 9.4;CaSO42H2O, 4.7;Na2HPO42H2O,752; KH2PO4,63.92; NH4Cl, 18.8and trace mineralssolution 0.47 ml:Na2EDTA, 2500;ZnSO47H2O, 100;MnCl26H2O, 30;H3BO3, 300;CoCl26H2O, 200;CuCl22H2O, 10;NiCl22H2O, 20;Na2Mo42H2O, 900;Na2SeO3, 20 andFeSO47H2O, 1000

Bioreactor [21]

ls and methods

atus

in advantage resulting from the microbial consor-d by acclimated activated sludge is the interactionl the species present in flocs. But, it is necessaryer things to pay attention to the phenomenon of

since, on the basis of mass transfer, flocs wouldcient than free cells in phenol degradation. Thatn why the acclimatization of microorganisms wasin a continuous bioreactor. The substrate is fed

ed at 12 L day1 with a peristaltic pump. Ultrafil-low fibre membranes (0.01m) immersed in themake possible to preserve a constant volume and

178 B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183

Fig. 1. Laborlow fibre modparmer instrummodel 7453-7

to preservtor.

An immthe cultivavolume of(10 g L1)air sourceflow rate omaintain thlevel (4.5 mnot to be li

For thewe have ussludge com

3.2. Analy

For thetank are filThe phenoCP 9001,[i.d. (32 mCourtaboeutor (FID). Pcurve madeleast duplicthe concensolved oxysured withmany). Theadjustmentsodium hydtrifuging atof sludge, acentrationsthe suspendcontains acshow bioloculty of ide

to make no attempt to separate SS into active and inactiveportions.

ynthetic efuent

sidering that the diversity of bacteria in activated sludgethem able to degrade most of the compounds, we have col-waste activated sludge from urban wastewater treatmentf Aix-en-Provence (France175,000 inhabitant equiva-hey are used as the source of microorganisms for phenolation in this study. The initial concentration near 3 g L1en increased to 10 g L1 by membrane filtration (hollowodule), under aeration.nuty (Ting tall csour

, Fe,e lesse) aperaa w

). Et, welt mH2P

ults

heno

edf glunol fenol Lrst 2aterring.5 gownatory scale bioreactor. (A) Bioreactor (90 L); (B) membrane (hol-ule); (C) air flowrate (010 L min1); (D) peristaltic pump (coleent model 7454-95); (E) peristaltic pump (cole parmer instrument

7); (F) feed medium (30 L); (G) permeate (20 L).

e the microorganisms themselves in the bioreac-

ersed membrane bioreactor (A) was employed fortion of organisms (Fig. 1). The reactor had a total90 L (with active volume of 60 L). The mixed liquorwas only agitated by aeration, from a compressedthrough a diffuser (H) in the bioreactors base at af 10 L min1 (C). The aeration rate was sufficient toe dissolved oxygen concentration near the saturatedgO2 L

1) during cultivation, so growth is assumedmited by oxygen.determination of the kinetics of phenol degradationed batch reactors (3 L) in which we put the activateding from reactor A.

tical methods

determination of phenol, samples from the aerationtered through filters having a pore size of 0.45m.l is analysed by gas chromatography (ChrompackMiddelburg, Netherlands) with a capillary columnm), length (15 m), film thickness (0.25m), SGE,f, France] and detected by a flame ionization detec-

3.3. S

Conmakeslectedplant olent). Tdegradhas befibre m

Theographaccordalmostgen, aZn, Cabecam(glucoing temchosen(C:N:PcontenThe sasalts: K

4. Res

4.1. P

Mixence o

of phe0.5 gphThe fiwastewand ducose (0was grhenol concentration is determined with a calibration

from known phenol standard. Each experiment is atated under identical conditions. Reproducibility of

tration measurements remains within 5%. The dis-gen concentration in the aeration tank is directly mea-a specific probe (Consort 932, Fisher Bioblock, Ger-pH is measured with pH meter Hanna Instrument. pH(pH 6.5) of the reactor content was performed withroxide. Suspended solids (SS) are measured by cen-3900 rpm (1900 RCF) for 15 min a sample of 25 mLnd by drying the deposit at 105 C for 24 h. The con-of the activated sludge are quantified as dry weight ofed solid. So, the determined mass of microorganismstive cells but also dead or inactive cells, which do notgical activity. However, because of the great diffi-ntifying this inactive portion of SS, we have decided

and then adperiod of 4ing this persystem wh

Table 2Mineral salt cand at the end

Parameters

C6H5OHNH4ClKH2PO4K2HPO4CaCl2Mg SO4rient medium was carried out according to the bibli-able 1). As seen before, there are various mediumso the type of microorganisms to be developed, but, inases there are a source of carbon, a source of nitro-ce of phosphorus, and oligoelements such as: Mg,Cu, Ni, K, Na and Co. However, phenol degradations efficient by increasing the concentrations of carbonnd nitrogen ((NH4)2SO4) sources (and with increas-ture) [15]. In order to prevent any deficiency we have

eight ratio of 100:5:1 for phenol:nitrogen:phosphorusffectively, as phenolics compounds are rich in carbon

have used phenol as a sole carbon source [18,22].edium was only composed by the following mineralO4, K2HPO4, NH4Cl, CaCl2, MgSO4 (Table 2).

and discussion

l acclimation and degradation

culture (activated sludge) was grown in the pres-cose and then adapted to increasing concentrationsrom an initial minimum inhibition concentration of1 + 0.4 gglucose L1 to the highest 3.0 gphenol L1.days the source of substrate is a mixture of urbanand glucose (0.25 gglucose L1); from the third day3 days we mixed phenol (0.46 gCOD L1) and glu-

COD L1). From the seventh day, the activated sludgein the presence of phenol as the sole carbon sourceapted to increasing concentrations of phenol over amonths; the reactor was continuously operated dur-

iod. The sludge was supposed to be acclimated to theen phenol was completely degraded in repeated uses

oncentrations in the activated sludge bioreactor at the beginningof the acclimation step

Initial (mg L1) Final (mg L1)1060 3030

200 57023 6929 85

7 2113 39

B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183 179

Fig. 2. Pheno(d) 0.7 g L1,

in fixed timand the pheof 3 g L1.

The kinassessed inphenol (Figwas taken(between 0working vostirrer wasthe mixingditions withevaluate thIt was obseduring theing do notsame initiaof phenol iconcentratimicroorganrate whenl degradation and variation of mixed liquor suspended solid in batch reactorinitia(e) 1.0 g L1 and (f) 1.5 g L1.

e intervals. At the end, the culture was acclimatednol concentration was maintained at a concentration

etics of phenol degradation by the sludge wasa batch reactor using different concentrations of

. 2). Sludge acclimated from the continuous processand phenol was added at different concentration.5 and 3 g L1) at the start of the experiment. Thelume of the reactor was maintained at 3 L, overhead

used to keep the organism in suspension and to ensureof air bubbles. Control experiments, in the same con-water containing phenol and substrate, were done to

e possible degree of phenol removal by gas transfer.rved that the phenol concentration remained constantexperiment, so we considered that aeration and mix-involve volatilization of phenol. Fig. 2 shows for thel cell concentration, that the higher the concentrations the more time it takes to be consumed. The phenolon in the culture medium decreased clearly when theisms started to grow. The acceleration of the growthphenol concentration decreased was characteristic

of a substrdegradationvaried as amedium.

Until ashow that tphenol fromcould not cbition effecabove the cteristic of abeen reportthe inlet phrespectivel

For the snol degradvaried as ature mediumexperimentto degradeup to 54 h fl phenol concentration: (a) 0.1 g L1, (b) 0.2 g L1, (c) 0.5 g L1,

ate inhibition phenomenon. The extent of phenoland the time required for phenol degradation

function of the initial phenol concentration in the

concentration of 2.5 g L1, the experimental resultshe mixed culture has a potential for the removal of

wastewaters. However, the activated sludge processope with phenol at loading rate in excess; the inhi-ts of phenol as substrate have become predominantoncentration of 3.5 g L1. This behavior is charac-toxic substrate metabolism. This kind of limit has

ed by Watanabe et al. [18] and Kibret et al. [21] whenenol loading was increased until 2.0 and 2.5 g L1,

y.ame initial biomass concentration, the extent of phe-ation and the time required for phenol degradationfunction of the initial phenol concentration in the cul-

. Degradation of more than 80% of phenol during alls occurred in less than 6 h. If one wants completelyphenol, the period should be prolonged, for exampleor an initial phenol concentration of 3 g L1.

180 B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183

Fig. 3. Effecbiodegradatio

The resuconcentratithat we havlaw.

This cuand its potinitial phenthe microo

4.2. Deterinhibition m

The kintions has bemodel thatdescriptionThis modelpresence ofthe metaboerogeneoussubstantialspecific grotype relatiodue to the iThe Haldathis inhibit

=KS +

with m thtration of sor the substerium forconcentratisubstrate in

The effect of a toxic compound on a treatment process isquantified in terms of the inhibition coefficient, Ki. It should be

that wono

te incan b

appium entlyhigh

on be

+ (Sical d

1m

+

is linkinroory the

KS

thereov

beon:

X

expohe en

(X2t of increased concentrations of phenol for the same degree ofn (80%) vs. the time taken for degradation.

lts shown in Fig. 3 indicate that an increase of phenolon affected the time of phenol removal (80%) it seemse an adequate estimation by the use of a exponential

rve shows the inhibiting character of this moleculeential biodegradadability. The more significant theol concentration is, the longer it will take time for

rganisms to degrade 80% of this phenol.

mination of kinetic parameterssubstrateodel

etics of phenol biodegradation by microbial popula-en largely studied [10,14,2326]. The mathematicalhas been found in order to get the statistically bestof the growth kinetics is the Haldane model (1965).describes relatively well the microbial growth in the

notedto the Msubstraeffect

Thebacterefficie

Atequati

=1

Graph(2):1

=

But thgrowth

Micelled b

dXdt

=

with XMo

kd mayequati

dXdt

=

In thefrom t

= lna substrate which is at the same time an inhibitor oflism of this microbial population that is pure or het-(mixed). Even at low concentrations, phenol had a

inhibitory effect on the specific growth rate (). Thewth rate tends to increase with the substrate (Monodnship), but rises to a peak and finally decreases

nhibitory effect of S as its concentration is increased.ne model that has frequently been used to describeion is:

mS

S + (S2/Ki) (1)

e maximum specific growth rate (h1), S the concen-ubstrate (mg L1), Ks the half-saturation coefficienttrate affinity constant (mg L1) (the affinity of a bac-a substrate) this constant is defined as the substrateon at which is equal to half max and Ki is thehibition coefficient (mg L1).

t2 In the literdation of pmixed cultuobtained bgiven in Ta

Since phHaldane eqdegradationprovided aauthors docentration.attemptingand S as inIt has beenmetabolic itunately, thof metabolcomplicatehen Ki is very large the Haldane equation simplifiesd equation (implies that the culture is less sensitive tohibition). So, low values ofKi show that the inhibitione observed at low phenol concentration.arent KS value is of practical importance because axpressing activity with a lower apparent KS value can

remove the pollutant down to lower concentration.er substrate concentrations, SKS, the Haldanecomes:

m

/Ki)(2)

etermination of Ki is obtained by linearization of Eq.

S

mKi(3)

earized Haldanes equation could not represent theetics [23].ganism growth rate in a batch reactor may be mod-following equation:

mS

+ S + (S2/Ki)X kdX (4)

concentration of biomass (mg L1).er, during exponential phase endogenous coefficientneglected. Eq. (4) therefore reduces to the following

(5)

nential phase, the specific growth rate is obtainedd of exponential growth phase and is calculated by:

/X1)t1

(6)

ature we found a lot of data concerning the degra-henol by P. putida and comparatively few data withre or acclimated activated sludge. Kinetic constants

y the Haldane model for phenol biodegradation areble 3.enol is an inhibitory substrate for most species, theuation has been frequently used to model phenol, and, compared to the Monod equation, has often

better representation of the observed data. But, manynot find the model suitable to the strong phenol con-Various kinetic relationships have been suggestedto describe the joint dependence ofonS as substratehibitor, for higher phenol concentration [24,26,27].determined that during phenol degradation various

ntermediates are produced and accumulated. Unfor-e Haldane model does not take into account the effectic intermediates on phenol degradation (model tood).

B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183 181

Table 3Kinetic constants for phenol biodegradation in batch reactor (Haldane equation)Culture T (C) pH [Phenol]maxa (mg L1) m 1 1 1

P. putida DSM 548 26 6.8 100 0.4P. putida CCRC14365 30 6.8 610 0.3P. Putida 800 0.9P. putida ATCC17514 0.8P. putida ATCC700007 30 7 200 0.0P. putida Q5 10 7 200 0.1P. putida MTCC1194 29.9 7.1 0.3P. uoroescens 0.8Acinetobacter + Pseudomonas 30 6.8 0.4Acinetobacter 30 350 0.8Trichosporon cutaneum R57 0.4Candida tropicalis 0.4Mixed culture 25 1450 0.1Mixed culture 15 40 0.2Mixed culture 28 6.6 900 0.2Mixed culture 20 6.8 0.3Mixed culture 0.7Mixed culture 0.1Mixed culture 0.4

a Represent

Althougadapted, wewhereas thon the concgrowth rateS0, has bee

The phetion and deas substrate

The threimental datLevenbergvals for miknown thatsitive to theparameterskinetic conmined on tdata.

Fig. 4. SpeciExperiment d

com

ratee gres gstang L

therludged toesuler, w

rderall

s utid quAmbient 6.5 2500

the maximum concentration being able to be degraded.

h some authors do not consider the Haldane modelhave calculated the specific growth rate using Eq. (1)

e dependence of activated sludge specific growth rateentration of phenol is shown in Fig. 4. The specific, , for each value of the initial phenol concentration,n determined in the exponential growth phase.nol degradation rate increases with phenol concentra-clines with further increases in phenol concentrationinhibition effects became important.

e-parameter model of Haldane was fitted to the exper-a using Statistica 6.0 software. We have worked on theMarquardt algorithm using 95% confidence inter-nimizing the sum of square of residuals. It is wellthe nonlinear optimization procedure is strongly sen-initial values and the variation intervals of the model

Thegrowththat thHaldanics con29.54 mwith ovated sexpectstrate rHowevsame o

The smspeciereache[4]. For this reason, the search for the values of thestants was constrained within boundaries predeter-he basis of the process knowledge and experimental

fic growth rate as a function of the initial phenol concentration.ata () and Haldane model ().

Comparedof Ki indicmild conceto substrateconcentratia concentraerature, ourIt is often dthat the Haphenol con0.5 and 1.0to be appardegradationsubstantiatet al. (Acinture) [26] wsubstrate inHowever inthis inadeqweaker than(h ) Ks (mg L ) Ki (mg L ) Reference36 6.19 54.1 [10]3 13.9 669 [14]0 6.93 284.3 [24]97 12.204 203.678 [28]51 18 430 [29]19 5.27 377 [30]05 36.33 129.79 [23]23 71.4 241 [25]18 29.37 370 [31]3 1.5 250 [27]2 110 380 [4]8 11.7 207.9 [32]43 87.44 107.06 [26]58 3.9 217 [2]60 25.4 173 [33]26 19.2 229.3 [34]46 53.9 516 [25]31 5.0 142 [35]38 29.5 72.4 This study

parison between experimentally obtained specific and the one that predicted by the model shows

owth kinetics of phenol could be represented byrowth kinetics model very well. The values of kinet-ts, m, Ks, Ki, obtained in this work as 0.438 h1,1 and 72.45 mg L1, respectively, are comparedpublished data in Table 3. As we have used acti-e, which is a mixture of many microorganisms, weobtain a possible competition for the common sub-

ting a lower growth rate, compared to a pure culture.e notice that we have a specific growth rate of theof magnitude as the one obtained for a pure culture.

magnitude of KS values indicates that for microbiallizing phenol, the maximum growth rate could beickly, if substrate inhibition has not been a factor.

to others culture studies (pure and mixed), the valueates that the inhibition effect can be observed in antration range. Mixed culture had a good resistanceinhibition. Thus, although we have a strong biomass

on (10 g L1) and that we inject into our batch reactortion in phenol much more significant than those in lit-s kinetics values are in the range of literature values.ifficult to determine the limits of a model; we guess

ldane model remains acceptable even with the strongcentrations. But, at substrate concentrations betweeng L1, the inadequacy of the Haldane model seems

ent. The Haldane model predicted shorter completestimes than these measured. The observations are

ed by other, like Wang and Loh (P. Putida) [24], Haoetobacter) [27] and Nuhoglu and Yalcin (Mixed cul-

ho have pointed out an inadequacy of the Haldanehibition kinetics at higher substrate concentrations.the case of a mixed culture and to our knowledge

uacy seems to be reached for phenol concentrationsin this study (for example 100 mg L1 for Ref. [26]).

182 B. Marrot et al. / Biochemical Engineering Journal 30 (2006) 174183

Unfortunately, in the literature most of the studies do not giveany information on microorganisms concentration in their reac-tor.

5. Conclu

A mixedin continuoexperimentcontainingvated sludgbioreactor

We knowporting thenecessary tulation whconsume iteasy to reainevitablymost favorsubject to rthe biodegtraditionaldangerous

From bacentration wHaldane eq(m = 0.43strate inhibbetween theters; Haldphenol conof the mixkinetic parstrate inhibvalidate thewith an extthen a real

Acknowled

Mexicanwork was s

Reference

[1] W. GernFernandphenolic

[2] G. Buitrlic compWater S

[3] A. Lantenols by36 (12)

[4] Z. Alexgrowth kneum RMicrob.

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95) 3HidalogicthropB. Pr

by Rked-bMarghighsts, RMordlow ccrob.

.G.ion bg. J. 6Gonznol i

idized.A.onas(3). Paonas

chem. Chudiateida sAnnausin

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radin711

Timuic coantaKumechol04) 1. Wanetics184uma

s. 31Nuhocess. Haooroph02) 7sion

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ernal MBR with initially the same synthetic substrateeffluent from an oil industry.

gments

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Biodegradation of high phenol concentration by activated sludge in an immersed membrane bioreactorIntroductionBiological treatmentAcclimated activated sludgeEffect of temperature and pH on phenol degradation

Materials and methodsApparatusAnalytical methodsSynthetic effluent

Results and discussionPhenol acclimation and degradationDetermination of kinetic parameters-substrate inhibition model

ConclusionAcknowledgmentsReferences