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International Biodeterioration & Biodegradation 63 (2009) 407–413

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Biodegradation of different molecular-mass polyphenols derivedfrom olive mill wastewaters by Geotrichum candidum

Nedra Asses a, Lamia Ayed a, Hassib Bouallagui a,*, Sami Sayadi b, Moktar Hamdi a

a Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Appliquees et de Technologie (INSAT), 2 Boulevard de la terre, B.P. 676, 1080 Tunis, Tunisieb Laboratoire des Bioprocedes, Centre de Biotechnologie de Sfax, BP: «K» 3038 Sfax, Tunisie

a r t i c l e i n f o

Article history:Received 4 August 2008Received in revised form30 October 2008Accepted 11 November 2008Available online 24 December 2008

Keywords:Geotrichum candidumOlive mill wastewaterPolyphenolsDecolourisationLignin peroxidaseManganese peroxidase

* Corresponding author. Tel.: þ21622524406; fax: þE-mail address: hassibbouallagui@yahoo.fr (H. Bou

0964-8305/$ – see front matter � 2008 Published bydoi:10.1016/j.ibiod.2008.11.005

a b s t r a c t

The decolourisation of fresh and stored olive mill wastewaters (OMW) and the biodegradation of threegroups (F1, F2 and F3) of phenolic compounds by Geotrichum candidum were investigated. Separatedphenolic compounds derived from natural OMW ultrafiltration using membranes with a cutoff 2and100 kDa. G. candidum growth on fresh OMW decreased pH and reduced COD and colour of 75% and 65%,respectively. However, on the stored-black OMW a failure of COD and colour removal were observed. G.candidum activity on this later substrate was enhanced by the addition of a carbon source easilymetabolised, misleading an improvement of the COD reduction and decolourization that reached 58%and 48%, respectively. Growth of G. candidum in the presence of F2 or F3 polyphenolic fractions inducedhigh decolourisation and depolymerisation of phenolic compounds. Whereas, very week decolourisationand biodegradation were observed with F1 fraction. Moreover, the highest levels of lignin peroxidase(LiP) and manganese peroxidase (MnP) activities were obtained in the presence of F2 fraction. Theseresults showed that increasing of molecular-mass of aromatics led to an increase in levels of depoly-merisation, decolourisation and COD removal by G. candidum culture.

� 2008 Published by Elsevier Ltd.

1. Introduction

The olive oil industry is very important in the Mediterraneancountries. The manufacture of olive oil yields two effluents, residualsolids (hask) and large quantities of liquid effluents (olive oilwastewaters). These effluents are composed of vegetation water ofolives plus washing and proceeding waters in addition to soft pulptune and oil in the form of a very stable emulsion. The annualproduction in the world is estimated to an amount over 107 m3

(Benitez et al., 1997).Typically, the weight composition of OMW is 83–96% water, 3.5–

15% organics, and 0.5–2% mineral salts. The maximum biologicaloxygen demand (BOD) and chemical oxygen demand (COD) reachconcentrations of 100 and 220 kg m�3, respectively. The organicfraction contains macromolecules, such as polysaccharides, lipidsand proteins, and a large phenolic content (Ehaliotis et al., 1999).

Phenolic compounds, which are usually present in OMW consist ofmonocyclic aromatic molecules, such as hydroxytyrosol, tyrosol,catechol, methylcatechol, Cafeic acid, and higher molecules masscompounds obtained through their polymerisation (Tziotzios et al.,2007). The last mentioned are recalcitrant to biodegradation.

216 71704329.allagui).

Elsevier Ltd.

Therefore, OMW cannot be released in the environment, but it shouldbe adequately treated before being discharged (Amaral et al., 2008).

One of the most promising OMW treatment technologies is theanaerobic biological digestion. However, the effectiveness of thistreatment is not always satisfactory, due to anaerobic microflorainhibition by OMW phenolic compounds which tend to persist inthe effluent of the treatment plant (Borja et al., 1995; McNamaraet al., 2008).

Aerobic biological methods seem to be especially suitable sincethe treatment leaves a residue of lower toxicity and diminishedphenol content. Fungal genera including Aspergillus niger (Hamdiand Ellouz, 1992; Borja et al., 1995; McNamara et al., 2008), Pha-nerochaete chrysosporium (Sayadi and Ellouz, 1992, 1995), G. can-didum (Garcia Garcia et al., 2000) and other which utilize a widerange of simple aromatic compounds and have high activities of therequisite catabolic enzyme, were proposed as an attractive alter-native for OMW pre-treatment. OMW remediation by means ofligniolytic fungi has been mostly addressed with an emphasis onthe identification of ligniolytic enzymes responsible for poly-phenols conversion and on the effects of operating conditions onenzyme secretion (Olivieri et al., 2006).

In our previous work, the ability of G. candidum to expressperoxidase enzymes and to decolourise OMW (Assas et al., 2000,2002; Ayed et al., 2005) was investigated. In fact, this fungusreduced significantly the colour and the COD of this effluent.

0

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1,5

2

2,5

3

3,5

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4,5

1 11 21 31 41 51 61 71 81 91 101 111 121 131 141Fractions

OD

at 280 n

m

1 2 3

Fig. 1. Changes in molecular-mass distribution of fresh OMW (-A-) and stored OMW(-,-). 1: Blue dextran (MM¼ 200 kDa), 2: Lysosym (MM¼ 15 kDa), 3: Syringic acid(MM¼ 198 Da).

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413408

The aim of this work is to study the ability of G. candidum tobiodegrade three groups of phenolic compounds derived fromnatural OMW through ultrafiltration technique, using membraneswith a cutoff 2 and 100 kDa. The decolourisation, the COD removal,and the effect of each fraction on the G. candidum growth, LiP andMnP activities as well as changes in molecular-mass distribution ofpolyphenols were investigated.

2. Material and methods

2.1. Olive mill wastewater

Olive mill wastewater used in this study was obtained from localthree-phase olive oil production plant (Tunis). OMW was centri-fuged at 6000� g for 30 min to eliminate solids and materials andstored at �20 �C. OMW from the same sample was also stored for 3month under ambient temperature and aerobic conditions. Maincharacteristics of raw OMW used in this work are shown in Table 1and Fig. 1.

2.2. OMW fractionation

Crude OMW was divided into different molecular-mass frac-tions by ultrafiltration (Gamma Filtration, France) through poly-sulphonate organic membranes (PCI, Laverstoke Mill, Whitchurch,England). These membranes have a multitubular configurationwith a cutoff 2 and 100 kDa. The molecular-mass fractions chosenwere low molecular-mass (F1<2 kDa), medium molecular-mass(2 kDa< F2<100 kDa) and high molecular-mass (F3>100 kDa).Main characteristics of fractions F1, F2 and F3 used in this workwere showed in Table 2 and Fig. 2.

2.3. Microorganism and culture procedures

G. candidum was isolated from OMW (Assas et al., 2000). Thisfungus was maintained through periodic transfer at 4 �C onSabauraud agar. Arthrospores were inoculated into Sabauraud agar,after 4 days at 30 �C, the surface of the slant was covered withwhite arthroconidia.

Cultures of G. candidum were conducted in 500 mL Erlenmeyerflasks containing 100 mL diluted and sterilized fresh or storedOMW. The centrifuged and sterilized OMW was diluted withdistilled water to 42 g COD L�1. The nitrogen source was ammo-nium sulphate (1 g L�1). The same amount of stored OMW withconcentration of 45 g COD L�1 was added with 10 g L�1 of glucoseas carbon source and 2 g L�1 of ammonium sulphate as nitrogensource. After inoculation with G. candidum arthroconidia suspen-sion at initial concentration of 105 arthroconidia mL�1, cultureswere incubated at 30 �C for six days. The initial pH was adjusted to6. All tests were conducted in triplicate.

Media used for the cultivation of G. candidum with OMW frac-tions (30%) contained per liter: glucose: 10 g; KH2PO4: 2 g;CaCl2$2H2O: 0.132 g; MgSO4$7H2O: 1.45 g; thiamine hydrochloride :1mg; D-diammonium tartrate: 1.2 mM; 20 mM, veratryl alcohol:0.4 mM and 20 mL of trace element solution. This trace elementsolution contained (per liter): FeSO4$7H2O, 1 g; MnSO4$4H2O, 1 g;

Table 1Characteristics of fresh and stored olive mill wastewater.

Characteristics Fresh olive mill wastewater Stored olive mill wastewater

pH 5.0� 0.2 5.6� 0.2COD (g L�1) 95� 2.2 68� 2.8Colour (A390 nm) 104.5� 2.5 150.5� 6.9TS (g L�1) 84.2� 1.5 59.3� 2.2TSS (g L�1) 14.9� 0.5 15.2� 0.3TVS (g L�1) 68.4� 1.2 53.4� 1.8

CuCl2, 0.025 g; CaCl2, 0.10 g; H3BO3, 0.056 g; ZnSO4$7H2O, 0.2 g;Na2MoO4$2H2O, 0.01 g.

This culture medium was buffered to pH 6 with cultures weregrown in a static conditions at 30 �C for six days. All cultures weregrown in triplicate.

2.4. Analytical methods

Growth represented by mycelium dry weight obtained bycentrifuging at 6000� g for 30 min, and the biomass was washedtwice with distilled water. The biomass evolution was estimated bymeasuring the dry weight after 24 h at 105 �C.

The analyses of pH, total solid, total volatile solids and totalsuspended solids (TS, TVS, TSS, COD; OMW decolourisation), werecarried out according to Standard Methods (APHA, 1998). The pHwas measured by means of a metrohm pH-meter model 632.

The soluble COD was measured on the centrifuged OMW at4000� g during 15 min using a PALINTEST 5000 photometer. OMWdecolourisation was assayed by measurement of the absorbance at390 nm using a CECIL-CE 1020 UV/VIS spectrophotometer.

2.5. Phenolic analysis

Phenol (with respect to gallic acid) concentrations were deter-mined spectrophotometrically according to the Folin–Ciocalteumethod (Garcia Garcia et al., 2000) using a CECIL-CE 1020 UV/VISspectrophotometer.

Phenolic compounds were prepared as follows: samples wereacidified with HCl (1 N) to pH 2 and extracted with ethyl acetate(10/30) at ambient temperature. The organic layer was combined

Table 2Characteristics of olive mill wastewater fractions F1, F2 and F3.

Characteristics F1 F2 F3

pH 4.64� 0.2 4.72� 0.2 4.86� 0.2COD (g L�1) 72� 2.2 107� 2.6 112� 4.8Molecular-mass F1<2 kDa kDa< F2<100 kDa F3>100 kDaPhenols (gallic acid g L�1) 2.18� 0.2 3.56� 0.3 1.77� 0.15

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0,6

0,8

1

1,2

1,4

1 9 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129Fractions

OD

at 280 n

m

1 2 3

Fig. 2. Changes in molecular-mass distribution of F1 fraction (-A-), F2 fraction (-,-)and F3 fraction (-:-). 1: Blue dextran (MM¼ 200 kDa), 2: Lysosym (MM¼ 15 kDa), 3:Syringic acid (MM¼ 198 Da).

0

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0,5

1 21 41 61 81 101 121

Fractions

Fractions

Fractions

OD

at 280 n

mO

D at 280 n

mO

D at 280 n

m

0

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1 12 23 34 45 56 67 78 89 100 111 122

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0,6

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1,6

1 10 19 28 37 46 55 64 73 82 91 100

b

c

a

Fig. 3. Changes in molecular-mass distribution of (a) untreated fresh OMW (-A-),treated fresh OMW (->-), (b) untreated stored OMW (---), treated stored OMW(-,-) and (c) untreated stored OMW (---) and treated stored OMW supplementedwith 10 g L�1 glucose and 2 g L�1 ammonium sulphate (-6-).

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413 409

with anhydrous Na2SO4 for 30 min. The extract was concentrated todryness in a rotary evaporator (at 45 �C) and redissolved witha mixture methanol/water (60:40). Total phenol content wasmeasured using the Folin–Ciocalteu’s phenol reagent (Merck),involving the successive additions of 5 mL sodium carbonate(200 g L�1) and 2.5 mL Folin–Ciocalteu’s phenol reagent to 50 mL ofproperly diluted sample. After 60 min at 20 �C the absorbance wasmeasured at 725 nm against distilled water.

2.6. Molecular-mass distribution of the polyphenolics

Gel filtration on Sephadex G-50 was used to analyze the polymericaromatic fraction present in different samples of OMW. Two millilitreof sample were filtered and placed on a Sephadex coarse G-50column (2.5� 60 cm) previously equilibrated with NaNO3 0.05 Mcontaining 0.02% sodium azide at a flow rate of 0.6 mL min�1. Theeffluent was collected on the basis of 3 mL per tube. The optic densityof these fractions was measured spectrometrically at 280 nm. Thecolumn was calibrated with syringic acid (MM¼ 198 Da), lysozym(MM¼ 15 kDa) and blue dextran (MM¼ 200 kDa).

2.7. HPLC analysis

A reversed-phase high performance liquid chromatographictechnique was developed to identify and quantify the major

Table 3Biomass, pH, COD and colour removals of fresh and stored OMW fermented for 6days with Geotrichum candidum as a function of culture conditions.

Diluted freshOMWþ 1 g L�1

(NH4)2SO4

Diluted storedOMWþ 1 g L�1

(NH4)2SO4

Diluted storedOMWþ 2 g L�1

(NH4)2SO4þ 10 g L�1 glucose

Biomass (g L�1) 4.8� 0.1 0.2� 0.0 3.2� 0.1pH 4.1� 0.2 7.8� 0.2 4.2� 0.1Initial COD (g L�1) 42.4� 2.2 45.2� 2.8 53.6� 4.1Final COD (g L�1) 10.6� 0.2 38.4� 3.7 22.5� 2.2COD removal (%) 75� 3.5 15� 2.5 58� 2,8Initial OD390 52.3� 3.3 110.6� 5.4 106.4� 4.2Final OD390 18.3� 1.2 168.4� 8.2 55.3� 3.2Colour removal (%) 65� 4.2 0 48� 3.2

phenolic compounds contained in the ethyl acetate extracts offresh, stored OMW and its different fractions. The HPLC chro-matograph was performed on a Shimadzu apparatus composed ofa LC-10 ATVP pump and a SPD-10 AVP detector. Elutes weredetected at 280 nm. The column was (4.6� 250 mm) model Shim-pach VP-ODS and its temperature was maintained at 40 �C. Thefollow rate was 0.5 mL min�1. The mobile phase used was 0.1%phosphoric acid in water (A) versus 70% acetonitrile in water (B) fora total running time for 40 min. The gradient was charged as follow:solvent B started at 20% and increased immediately to 50% in30 min. After that, elution was conducted in the isocratic modewith 50% solvent B within 5 min. Finally, solvent B decreased to 20%until the end of running.

Table 4pH, Biomass, COD and colour removals of OMW fractions F1, F2 and F3 fermented for6 days with G. candidum.

F1 F2 F3

Biomass (g L�1) 2.3� 0.1 4.2� 0.2 5.4� 0.2pH 4.5� 0.2 4.8� 0.2 4.2� 0.1Initial COD (g L�1) 28.6� 1.4 39.1� 2.5 40.6� 3.6Final COD (g L�1) 21.8� 1.6 17.9� 0.9 23.6� 1.9COD removal (%) 23.6� 1.6 54.2� 2.4 41.9� 2.1Initial OD390 16.5� 1.2 31.3� 2.1 44.9� 4.6Final OD390 10.4� 0.6 10.6� 0.2 20.7� 1.3Colour removal (%) 37.1� 2.2 66.2� 3.6 53.9� 1.9

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413410

2.8. Lignin peroxidase activity

Lignin peroxidase (LiP) activity was determined using veratrylalcohol as substrate (Tien and Kirk, 1984).The assay mixture con-tained 2 mM veratryl alcohol and 0.4 mM H2O2 in 50 mM sodiumtartrate buffer, pH 2.5 Oxidation of veratryl alcohol was followed bymeasuring the increase in absorbance at 310 nm because of theformation of veratraldehyde from (£310¼ 9300 M�1 cm�1). Enzymeactivity was expressed in international units (IU).

2.9. Manganese peroxidase activity

Manganese peroxidase (MnP) activity was determined usingMnSO4 as substrate (Giardina et al., 2000). The assay mixturecontained 0.5 mM MnSO4 and 0.5 mM H2O2 in 50 mM sodiummalonate buffer, pH 4.5 Oxidation of Mn2þ was followed bymeasuring the increase in absorbance at 270 nm due to theformation of Mn3þ-malonate from (£270¼11590 M�1 cm�1).Enzyme activity was expressed in international units (IU).

3. Results

3.1. Biodegradation of fresh and stored OMW by G. candidum

Table 1 shows the main characteristics of fresh and stored OMW.The pH of fresh OMW is lower than that of stored OMW. The colour

Table 5HPLC evaluation of major phenolic compound identified in OMW and in different fractio

Phenolic monomers (mg L�1) Fresh OMW (mg L�1) Stored OMW (mg L�1)

Untreated Treated Untreated Treated

Hydroxytyrosol 3450.7 613.5 930.5 183.5Tyrosol 439.4 114.5 115.9 32.43,4-Dihydroxyphenyl acetic acid 67.1 12.4 174.5 77.5Vanillic acid 191 6.1 – –Cafeic acid 2.6 0.63 35.8 32.4Coumaric acid 0.8 0.3 15.5 1.6Syringic acid – – – –Ferulic acid 1.7 1.5 96.7 33.6

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6 7 8Time (days)

LiP

(U

L

-1)

a

Fig. 4. Time course of (a) lignin peroxidase and (b) manganese peroxidase activities du

and the phenols content increased after some months, which maybe attributed to the polymerisation of phenolic compounds withlow molecular-masses. Gel-filtration chromatography of untreatedfresh and stored OMW showed two families of aromatics, the firstone with high molecular-mass and the second one with lowmolecular-mass. The first major peak in stored OMW presentsdarkly coloured polymers, which was resulted from the polymeri-sation and auto-oxidation of simple phenolic compounds (Fig. 1).

As shown in Table 3 cultures of G. candidum on centrifuged anddiluted fresh OMW decreased pH and reduced COD and colour by75% and 65%, respectively after 6 days of incubation. When G.candidum was incubated with diluted stored OMW as a sole carbonsource, growth was inhibited compared with diluted fresh OMW.Moreover, an increase of pH and colour was observed due to phenolpolymerisation reactions. However the addition of 10 g L�1 ofglucose as easily metabolised carbon source, improved the COD andcolour removal yields that reached 58% and 48%, respectively.

Gel-filtration chromatography was performed on the liquidphase sampled at the beginning and the end of incubation. Withfresh OMW, results (Fig. 3a) indicated that the molecular-massdistribution of polyphenols changes after 6 days of incubation. Thehigh molecular-mass polyphenols (� 60 kDa) fraction becameprogressively more populated at the expenses of the low molec-ular-mass fraction. With stored-black OMW, decolourisation wasnot observed. In fact, gel filtration on Sephadex analyses ofphenolics compounds showed the polymerisation of phenoliccompounds into higher molecular-mass compounds (Fig. 3b). Theelution patterns of untreated and treated stored OMW supple-mented with glucose after 6 days of incubation showed animportant reduction in molecular-mass distribution of polyphenols(Fig. 3c).

HPLC analysis of the final extract obtained from fresh and storedOMW at the beginning and the end of incubation revealed thepresence of several phenolic compounds as shown in Table 5.Hydroxytyrosol and tyrosol were the major compounds detected.Their concentrations were 3450 and 439 mg L�1 respectively infresh OMW and 930 and 110 mg L�1 respectively in stored OMW.3,4-dihydroxyphenyl acetic acid, vanillic acid, cafeic acid, and

ns F1, F2 and F3.

F1 fraction (mg L�1) F2 fraction (mg L�1) F3 fraction (mg L�1)

Untreated Treated Untreated Treated Untreated Treated

393 221 1484 1102 318 29075 67 262 248 178 16912 23 45 61 40 6417 9 49 6 25 4– – – – – –4 7 7 13 870.8 1.4 0.5 1.7 0.5 0.7– – – – – –

0 1 2 3 4 5 6 7 8Time (days)

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140

Mn

P (U

L

-1)

b

ring fermentation of fresh (->-) and stored (-,-) OMW by Geotrichum candidum.

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6

1 15 29 43 57 71 85 99 113 127 141Fractions

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at 280 n

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0

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6

OD

at 280 n

m

0

1

2

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6

OD

at 280 n

m

1 14 27 40 53 66 79 92 105 118 131Fractions

Fractions

1 16 31 46 61 76 91 106 121 136 151

a

b

c

Fig. 5. Changes in molecular-mass distribution of (a) untreated F1 (-A-) and treatedF1 (->-), (b) untreated F2 (---) and treated F2 (-,-) and (c) untreated F3 (---) andtreated F3 (-6-).

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413 411

coumaric acid were present at lower concentrations. Treated freshOMW and stored OMW supplemented with glucose showeda significant decrease of the concentration of total simple phenol(Table 5).

Furthermore, the time courses of LiP and MnP activity by G.candidum growth on all these mediums were analysed (Fig. 4). LiPand MnP activity showed a maximum activity in the 5th day andthen decreased. Maximum activity was obtained in the mediumcontaining stored-black OMW supplemented with 10 g L�1 ofglucose and 2 g L�1 of ammonium sulphate.

3.2. Biodegradation of the OMW isolated fractions by G. candidum

The toxicity of OMW is mainly due to its phenolic or polyphenoliccompounds. The molecular-mass of these polyphenolics varied fromthe simple phenolics to the polymers of molecular-mass whichexceed 100 kDa. The elution patterns of the three fractions F1, F2 andF3 were examined by gel filtration in order to determine the distri-bution of phenolics (Fig. 2). Untreated F1 fraction showed one familyof aromatic compounds which could correspond to simple pheno-lics, o-diphenols and monomeric flavoids in a first group. Whereas,F2 fraction had a hydrolysable tannins, in the second group. F3contained complex and darkly coloured polyphenols such as ‘‘humicacid like’’, condensed tannins and anthocyanes. HPLC analysisrevealed the presence of several monomeric compounds withdifferent concentration as shown in Table 5.

The decolourisation and COD removal of the three molecular-mass polyphenolic fractions by G. candidum were examined. The F2fraction was decolourised at a high percentage. In this case,decolourisation reached more than 66% after 6 days. The decol-ourisation of the F3 fraction was lower and the overall decolour-isation after 6 days was about 54%. However, the decolourisation ofthe F1 aromatic fraction exhibited low levels, which did not exceed37% (Table 4). The percentage of COD removal decreased from 54%for F2 to less than 42% for the F3 fraction. However, the CODremoval was lower for the F1 aromatic fraction, which did notexceed 23% after 6 days of treatment (Table 4).

The elution patterns of the three fractionated OMW untreatedand treated with G. candidum showed a significant depolymerisa-tion and/or degradation of F2 fraction by G. candidum (Fig. 5b). Itwas reduced to a peak corresponding to aromatics with interme-diate molecular-mass.

An extensive reduction in the molecular-mass distribution ofpolyphenolics was particularly observed in the treated F3 fraction,where a depolymerisation of high molecular-mass polyphenolicswas occurred (Fig. 5c). In contrast to F2 and F3 fractions for whichpart of aromatics were consumed by the fungus, the F1 fraction wasrecalcitrant and only slight bioconvertion of the low molecular-mass aromatics was observed. However, a very weak depolymer-isation of F1<2 kDa was observed (Fig. 5a). In fact, increasing ofmolecular-mass of aromatics led to an increase in the depolymer-isation levels and COD removal by G. candidum cultures. HPLCanalysis showed a significant removal in the concentration of totalsimple phenol content in treated fractions F1, F2 and F3 (Table 5).

The results presented in Fig. 6 confirmed the role of LiP and MnPin the decolourisation of OMW fractions. Maximum activity wasobtained in the medium containing the F2 aromatic fraction.

4. Discussion

G. candidum grew on fresh OMW metabolising sugars and othersimple compounds and then reducing COD, showed a greatercolour removal. In this condition, an important decrease of pH wasobserved from 6 to 4.1, due to the consumption of sugar content.Nevertheless, pH value remained favourable to enzymatic decol-ourisation as reported by Kim and Shoda (1999). These results arein agreement with Fadil et al. (2003), they have performed theeffectiveness of Geotrichum sp. for the bioremediation of OMW inagitated culture and they reported a reduction of COD and phenoliccompounds of 55 and 46.6% respectively.

0

20

40

60

80

100

120

LiP

(U

L

-1)

0

20

40

60

80

100

120

Mn

P (U

L

-1)

0 1 2 3 4 5 6 7 8Time (days)

0 1 2 3 4 5 6 7 8Time (days)

ba

Fig. 6. Time course of (a) lignin peroxidase and (b) manganese peroxidase activities during fermentation of F1 fraction (-�-), F2 fraction (-þ-) and F3 fraction (-B-) by G. candidum.

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413412

The high OMW decolourisation was correlated with theproduction of extracellular peroxydases which was purified andcharacterised by Kim and Shoda (1999). The most notable reductionin all parameters analyzed occurred during the first 6 days showedthat LiP and MnP enzymes have a key role in the OMW degradationprocess. It has been reported that ligninolytic enzymes are involvedin OMW degrading process (Martirani et al., 1996; Sayadi et al.,2000). Moreover G. candidum has the ability to decolourise OMWfurther to the production of lignin peroxidase (Ayed et al., 2005).

Practically, no biodegradation was achieved when stored OMWwas not supplemented with nutrients. The COD and colour removalyield of the media containing only stored OMW were very lowthan those of the media supplemented with 10 g L�1 of glucose ascarbon source and 2 g L�1 of ammonium sulphate as nitrogensource. When G. candidumwas incubated with stored oxidised OMWas a sole carbon source, the growth was rapidly inhibited and thecolour increased because of phenol polymerisation. Olivieri et al.(2006) suggested the relative importance of polymerisation versuspreferential mineralisation of the low molecular-mass phenoliccompounds as the relevant pathway to polyphenols bioconversion.However, glucose addition as a carbon source improved CODremoval and induced the decolourisation. In fact, stored OMW is noteasily biodegradable by G. candidum because this fungus requiressugar to express the decolourisation ability as mentioned by Assaset al. (2002) and Ayed et al. (2005). Similar results have beenreported concerning the role of glucose in fungal decolourisation ofcotton bleaching with P. chrysosporium (Fu-Ying et al., 1999). Ourresults suggest that culture conditions that yield high LiP and MnPactivity lead to higher levels of OMW decolourisation by G. candidumdue to depolymerisation of phenolic compounds with high molec-ular-mass which are the most recalcitrant compounds.

The treatment of different molecular-mass polyphenolcompounds (F1, F2 and F3), derived from OMW by G. candidumshowed low levels of F1 aromatic fraction decolourisation. Themaximum degree of polyphenols bioconversion was observed in F2and F3 under aerobic operating culture of G. candidum. The depo-lymerisation of polyphenols with a molecular-mass higher than byG. candidum was observed. These compounds did not affect thegrowth of this fungus, which was accompanied by the production ofLiP and MnP enzymes. A resistance of low molecular-mass phenolicto degradation by G. candidum was observed. These compounds didnot affect the growth of this fungus but rather its degradativesystem. However, F1 fraction is well degraded by aerobic bacteria.Sayadi et al. (2000) suggested that biodegradation of high concen-tration of simple and low molecular-mass phenolic compoundscould be achieved by new isolated or genetically-engineeredbacteria under optimized aerobic process technology. By contrasthigh molecular-mass polyphenols are degraded particularly bywhite rot fungi, process requiring longer treatment periods. Thepresent results showed that G. candidum, one of the best OMW

degrader, can attack OMW and especially the high molecular-masswhich led to a decrease in colour and COD removal efficiencies.

Results of the present study are encouraging in the perspectiveof the development of an integrated OMW remediation process.This issue is represented by proper control of the stoichiometry ofpolyphenols bioconversion by G. candidum. The possibility tofavour polyphenols conversion by supplying additional nutrients isdemonstrated in the present study. It remains to be assessedwhether the addition of some cheap and easily available nutrientsto OMW might be conveniently accomplished in practice, also inthe light of its impact on process economics.

Acknowledgements

The authors wish to acknowledge the Ministry of SuperiorEducation and Scientific Research and Technology, which hasfacilitated the carried work.

References

Amaral, C., Lucas, M.S., Coutinho, J., Crespi, A.L., Anjos, M.R., Pais, C., 2008. Micro-biological and physicochemical characterization of olive mill wastewaters froma continuous olive mill in Northeastern Portugal. Bioresource Technology 99,7215–7223.

APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20thed. American Public Health Association, Washington, DC, USA,.

Assas, N., Ayed, L., Marouni, L., Hamdi, M., 2002. Decolorization of fresh and storedblack olive mill wastewaters by Geotrichum candidum. Process Biochemistry 38,361–365.

Assas, N., Marouani, L., Hamdi, M., 2000. Scale down and optimization of olive millwestewaters decolorization by Geotrichum candidum. Bioprocess Engineering22, 503–507.

Ayed, L., Assas, N., Sayadi, S., Hamdi, M., 2005. Involvement of lignin peroxidase inthe decolourisation of black olive mill wastewaters by Geotrichum candidum.Letters in Applied Microbiology 40, 7–11.

Benitez, J., Beltran-Heredia, J., Torregrosa, J., Acero, J.L., Cercas, V., 1997. Aerobicdegradation of olive mill wastewaters. Applied Microbiology and Biotechnology47, 185–188.

Borja, R., Martin, A., Alonso, V., Garcia, I., Banks, C.J., 1995. Influence of differentaerobic pre-treatment on the kinetics of anaerobic digestion of olive millwastewater. Water Research 19, 489–495.

Ehaliotis, C., Papadopoulou, K., Kotsou, M., Mari, I., Balis, C., 1999. Adaptation andpopulation dynamics of Azotobacter vinelandii during aerobic biological treat-ment of olive mill wastewater. FEMS Microbiology Ecology 30, 301–311.

Fadil, K., Chahlaoui, A., Ouahbi, A., Zaid, A., Borja, R., 2003. Aerobic biodegradationand detoxification of wastewaters from the olive mill industry. InternationalBiodeterioration and Biodegradation 51, 37–41.

Fu-Ying, Z., Jeremy, S.K., Kelvin, N.T., 1999. Decolourisation of cotton bleachingeffluent with wood rotting fungus. Water Research 33, 919–928.

Garcia Garcia, I., Jimenez Pena, P.R., Bonilla Venceslada, J.L., Martin Martin, A., MartinSantos, A.A., Ramos Gomez, E., 2000. Removal of phenol compounds from olive oilmill wastewater using Phanerochaete chrysosporium, Aspergillus niger, Aspergillustereus and Geotrichum candidum. Process Biochemistry 35, 751–758.

Giardina, P., Palmieri, G., Fontanella, B., Rivieccio, V., Sannia, G., 2000. Manganeseperoxidase isoenzymes produced by Pleurotus ostreatus grown on woodsawdust. Biochimica et Biophysica Acta 376, 171–179.

Hamdi, M., Ellouz, R., 1992. Bubble column fermentation of olive mill wastewaters byAspergillus niger. Journal of Chemical Technology and Biotechnology 54, 331–335.

N. Asses et al. / International Biodeterioration & Biodegradation 63 (2009) 407–413 413

Kim, J., Shoda, M., 1999. Purification and characterization of a novel peroxidase fromGeotrichum candidum Dec 1 involved in decolourization of dyes. Applied andEnvironmental Microbiology 65, 1029–1035.

Martirani, L., Giardina, P., Marzullo, M., Sannia, G., 1996. Reduction of phenolcontent and toxicity on olive oil mill waste waters with the lignolytic fungusPleurotus ostreatus. Water Research 30, 1914–1918.

McNamara, C.J., Anastasioy, C.C., O’Flaherty, V., Mitchell, R., 2008. Bioremediation of olivemill wastewater. International Biodeterioration and Biodegradation 61, 127–134.

Olivieri, G., Marzocchella, A., Salatino, P., Giardina, P., Cennamo, C., Sannia, G., 2006.Olive mill wastewater remediation by means of Pleurotus ostreatus. BiochemicalEngineering Journal 31, 180–187.

Sayadi, S., Ellouz, R., 1992. Decolourization of olive mill wastewater by the white rotfungus Phanerochaete chrysosporium: involvement of the lignin degradingsystem. Applied Microbiology and Biotechnology 37, 813–817.

Sayadi, S., Ellouz, R., 1995. Roles of lignin peroxidase and manganeseperoxidase from phanerochaete chrysosporium in the decolorization of olivemill wastewaters. Applied and Environmental Microbiology 61 (3),1098–1103.

Sayadi, S., Allouche, N., Jaoua, M., 2000. Detrimental effects high molecular-massepolyphenols on olive mill wastewater biotreatment. Process Biochemistry 35(7), 725–735.

Tien, M., Kirk, T.K., 1984. Lignin degradation enzyme from Phanerochaete chrys-osporium: purification, characterization and catalytic cycle of a unique H2O2-requiring oxygenises. Proceedings of the National Academy of Sciences USA 81,2280–2284.

Tziotzios, G., Michailakis, S., Vayenas, D.V., 2007. Aerobic biological treatment ofolive mill wastewater by olive pulp bacteria. International Biodeterioration andBiodegradation 60, 209–214.