B

A

Ttctamtb©

K

1

ppeaic

itgadc

1d

ARTICLE IN PRESS+ModelEJ-4418; No. of Pages 6

Biochemical Engineering Journal xxx (2007) xxx–xxx

Decolorization of semisolid olive residues of “alperujo” during the solidstate fermentation by Phanerochaete chrysosporium, Trametes versicolor,

Pycnoporus cinnabarinus and Aspergillus niger

F. Aloui a, N. Abid a, S. Roussos b, S. Sayadi a,∗a Laboratoire des Bioprocedes, Centre de Biotechnologie de Sfax, B. P. “K”, 3038 Sfax, Tunisia

b 2 Institut de Recherche pour le Developpement (IRD), Unite 185 Biotrans, IMEP D42, Faculte des Sciences et Techniques,Universite Paul Cezanne, 52, A. Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France

Received 4 January 2006; received in revised form 20 November 2006; accepted 11 January 2007

bstract

Studies were carried out on decolorization of semisolid olive mill residues called “alperujo” (AL), by four strains of Phanerochaete chrysosporium,rametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger in solid state frementation (SSF). Fungal strains were selected by their abilityo grow on olive mill waste water. The treatment of AL with P. chrysosporium led to higher removal of organic matter and decolorization than P.innabarinus and T. versicolor. The strain A. niger leads to a relative improvement of the biodegradability of OMW. Using P. chrysosporium, thereatment of AL substrate in SSF showed an efficient decolorization and an appreciable COD and phenolic content removal only in the presence of

support. Sugarcane bagasse used as support improved the oxygen transfer in the culture. The extracellular fluid of P. chrysosporium (composedainly of LiP) exhibited high ability to decolorize AL showing the efficiency of the enzyme produced. A percentage of sugarcane bagasse equal

o 30% was an optimal condition to improve the growth and the decolorization of AL. The results showed good prospects of using the threeasidiomycetes, in particular P. chrysosporium, for the Decolorization of AL.

2007 Elsevier B.V. All rights reserved.

etry;

cPr

“pdgnlh(o

eywords: Alperujo; Decolorization; Phanerochaete chrysosporium; Respirom

. Introduction

Pollution by olive mill waste (OMW) is becoming a crucialroblem in the Mediterranean area, particularly for the mainroducers of oil, Italy, Spain, Greece and Tunisia. This efflu-nt is black and highly toxic owing to its high concentration ofromatic compounds. The chemical composition of this wastencludes polyphenols such as tannins, anthochyanins, and cate-hin [1].

Several studies using microorganisms possessing the abil-ty to degrade phenolic compounds have been carried out inhe last years. Phanerochaete chrysosporium, a white rot fun-us, secretes the extracellular enzymes, lignin peroxidases (LiP)

Please cite this article in press as: F. Aloui et al., Decolorization of setation by Phanerochaete chrysosporium, Trametes versicolor, Pycnopodoi:10.1016/j.bej.2007.01.005

nd manganese dependent peroxidases (MnP). These enzymesegrade lignin and an extremely diverse range of aromaticompounds such as polycyclic aromatic hydrocarbons [2], and

∗ Corresponding author. Tel.: +216 74 440 452; fax: +216 74 440 452.E-mail addresses: sami.sayadi@cbs.rnrt.tn, ssayadi@arrakis.es (S. Sayadi).

atiGsfp

369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2007.01.005

Solid state fermentation

hlorophenols [3]. The wide specificity of these enzymes made. chrysosporium a good candidate to eliminate xenobiotic andecalcitrant compounds.

Two processes are used for the extraction of olive oil: thethree-phase” process and the “two-phase” process. The two-hase centrifugation system for oil extraction was developeduring the nineties. This system greatly reduces wastewatereneration and its contaminated load, but it still produces aew by-product called “alperujo” (AL), a solid material ofow consistency, whose main agrochemical characteristicsave been extensively reported [4]. Solid state fermentationSSF) is defined as any fermentation process carried outn a solid material (employing either a natural support orn inert support) in absence of free flowing liquid. SSFechnique is known to offer many advantages of commercialmportance, as compared to submerged fermentations [5].

misolid olive residues of “alperujo” during the solid state fermen-rus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),

utierrez-Sanchez et al. [6] showed that the solid state cultureeems to be a better system for caffeine degradation. It wasound to be able to overcome catabolic repressions, to endroduct inhibition and to tolerate high metal salt concentrations

IN+ModelB

2 ineer

[o

mmaiTalcods

bafCss

2

2

H(em

W

M

rk0Cpan

oc

as

uceveaa

2

t

tscfsodpspa(

2

iTTpvdAcCor(

ai

awlte

2

dtaac[

ARTICLEEJ-4418; No. of Pages 6

F. Aloui et al. / Biochemical Eng

7]. Consequently, SSF could be used in the detoxificationf AL.

Since white rot fungi detoxify (OMW) and lignocelluloseaterials, it would seem feasible that a WRF solid state fer-entation of AL might also detoxify this substrate. The intense

dherence of filamentous mushrooms to the substrate makes itmpossible to evaluate the biomass by the classical methods [8].he respirometry is a solution. It consists in monitoring andnalysing the gases involved in the process to observe the evo-ution of mycelium growth by an indirect manner. Contents inarbon dioxide and in oxygen, products of SSF, are the resultsf the microbial activity [9]. These analyses permit to follow theifferent phases of culture without disrupting the fermentationystem [10].

This work tested the treatability of AL by SSF. It used threeasidiomycetes known by their ability to degrade lignin andstrain of Aspergillus (An10), which showed a high biomass

ormation in SSF. This treatment was conducted by measuringO2 and O2 concentrations in exhaust gases, using an automatic

ampler connected to a gas chromatograph and a data acquisitionystem [10].

. Materials and methods

.1. Strains and culture conditions

The strain used in this study was: P. chrysosporiumD, a monoconidiosporous isolate from strain BKM-F-1767

ATCC 24725). It was maintained at 4 ◦C on 2% maltxtract broth slants. Subcultures were routinely made every 2onths.Trametes versicolor and Pycnoporus cinnabarinus were 2

RF produced in the Centre de Biotechnologie de Sfax, Tunisia.A. niger (An10) was kindly provided by the Laboratory of

icrobiology, IRD de Marseille, France.The basal medium used for the cultivation of P. chrysospo-

ium, T. versicolor and P. cinnabarinus, contained (perg): KH2PO4: 1 g; CaCl2·2H2O: 0.07 g; MgSO4·7H2O:.35 g; FeSO4·7H2O: 0.035 g; ZnSO4·7H2O: 0.023 g anduSO4·5H2O:0.0035 g. This culture medium was buffered toH 6.5 with di-sodium-tartrate (20 mM). Veratryl alcohol wasdded to 0.4 mM. The carbon source was glycerol (10 g l−1). Theitrogen source was ammonium tartrate at 20 mM of nitrogen.

The substrate used for this work (Alperujo) was from an oliveil manufacture (using the “two-phase” system) located in theity of Sfax (Tunisia).

Sugarcane bagasse, with 0.8–2.0 mm particle diameter rangend exhaustively washed with distilled water, was used as aupport and sterilized by steaming for 20 min [10].

The inoculated moist medium was transferred to a glass col-mn fermentor (4 cm in diameter and 20 cm in height). Allolumn fermentors were placed in a water bath as describedlsewhere by Raimbault and Alazard [11]. Experiments with T.

Please cite this article in press as: F. Aloui et al., Decolorization of setation by Phanerochaete chrysosporium, Trametes versicolor, Pycnopodoi:10.1016/j.bej.2007.01.005

ersicolor and P. cinnabarinus were carried out at 25 ◦C whilexperiments with P. chrysosporium and A. niger were carried outt 37 ◦C. Humidified air passed through each column fermentort a rate of 50 ml min−1.

poqc

PRESSing Journal xxx (2007) xxx–xxx

.2. Samples treatment

The content of each incubator was weighed and homogenizedo obtain samples for analysis.

To study the parameters of fermentation in the course of theime, it was necessary to determine a complete column corre-ponding to one time of the fermentation. The content of theolumn was weighed in the beginning and at the end of theermentation in order to calculate losses in dry weight of theubstrate (LW). A sample of 5–10 g served to the determinationf the moisture content and the dry matter (DM). Analyticaleterminations were carried out on the water extract of the sus-ension of 5 g of fresh material in 45 ml of distilled water, aftertirring. The pH and electrical conductivity (EC) of the sus-ension were measured directly after agitation. For the ulteriornalyses, it was necessary to separate the cheek by centrifugation10,000 rpm min−1).

.3. Analytical methods

Dry weight and moisture content were determined by weigh-ng samples before and after drying overnight at 105 ◦C. Ash andVS were analysed, by loss on ignition at 600 ◦C for 2 h. Theotal nitrogen content (TN) was analysed as Kjeldahl—N. Therotein content was calculated by multiplying TN by the con-ersion factor of 6.25. Water soluble phenolic substances wereetermined on the aquous methanol extract of each sample ofL after shaking for 2 h in a mechanical shaker. Total phenol

oncentrations were quantified by the colometric method [12].OD was estimated as described by Knechtel [13]. Biologicalxygen demand (BOD5) was determined by the manomet-ic method with a respirometer [BSB-Controller Model 620TWTW)].

The decolorization was determined by the measurement ofbsorbance at 395 nm. Results were expressed in comparison tonitial samples in the same conditions [14].

Lignin peroxidase activity was determined using the veratryllcohol oxidation assay [15]. Manganese-dependent peroxidaseas assayed according to Paszcynski et al. [16], using vanil-

ylacetone as substrate. Laccases were determined accordingo the method of Szklarz et al. [17]. Enzymatic activities werexpressed in international units (U).

All the analyses were made in triplicate.

.4. Solid state fermentation system (SSF)

The design and control of SSF system was previouslyescribed by Saucedo-Castaneda et al. [10]. The column reac-ors were incubated at 25 ◦C or 37 ◦C, and aerated (50 ml min−1)fter humidification of the air. During SSF, the gas produced wasnalysed automatically by an online automated monitoring andontrol system using a CPG system for O2 and CO2 analysis10]. The obtained data were used to establish kinetic growth

misolid olive residues of “alperujo” during the solid state fermen-rus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),

rofiles through the model systems based on the O2 uptake rater the CO2 production rate and also to calculate the respiratoryuotient (RQ), defined as the ratio of CO2 produced and O2onsumed [18].

IN PRESS+ModelB

ineering Journal xxx (2007) xxx–xxx 3

3

3c

sbfsWw

moofioDrgt0

atdsAr

oTgcwmlio

ttoi

Table 1Main characteristics of the used “alperujo” (dry weight)

Parameters Mean

Moisture (%fresh weight) 65.0pH 4.82EC (dS m−1) 3.58COD (g kg−1) 237BOD (g kg−1) 92.4Total solid (%fresh weight) 35.0Ash (%fresh weight) 7.4TSS (%fresh weight) 35.0TVS (%fresh weight) 32.4TN (g kg−1) 10.8Protein (g kg−1) 67.5Phenolic content (g kg−1) 18.6

TPp

S

FLFFRFR

ARTICLEEJ-4418; No. of Pages 6

F. Aloui et al. / Biochemical Eng

. Results and discussion

.1. Comparison of P. chrysosporium to other strains inulture on the AL substrate in SSF

The aim of the present work was to study the capability ofome microorganisms to treat AL of OMW and their kineticehaviour in comparison to several other microorganisms usedor the same purpose. The first objective was to select an effectivetrain for the decolorization of AL substrate. Three cultures of

RF were screened for their ability to degrade lignin. A. nigeras selected for its ability to grow in OMW.Indeed, to prove this, we adopted the measurement of

ycelium apical growth as a good indication of the specificityf each mushroom. Similarly, this parameter served in previ-us works to indicate the invasion of a solid state medium bylamentous fungi [19]. Thus, in this study, the measurementf mycelium apical growth in the nutritional media (Potatoesextrose Agar) showed that A. niger had the fastest growth

ate (4.8 mm h−1) of the three basidiomycetes. However, therowth rate of P. chrysosporium (2.02 mm h−1) was faster thanhose of T. versicolor and P. cinnabarinus (0.36 mm h−1 and.15 mm h−1, respectively).

Chemical characterization showed that used AL (Table 1) hashigh moisture content, slightly acidic pH values and a high con-

ent of organic matter, especially phenolic compounds. AL wasemonstrated to be a powerful source of phenolic compounds-pecifically, 8680 �g g−1 [20]. The ration COD/BOD of the usedL, estimated to 2.6, show a possible biodegradation of this

esidue.A kinetic and enzymatic study was carried out on AL mixed

r not with sugarcane bagasse degradation by fungi, under SSF.able 2 shows the inhibitory effect of the substrate on therowth of the four strains, especially in the case of T. versi-olor where no growth was detected (Fig. 1a). A slight growthas observed for P. chrysosporium, without decolorizing theedium. Besides, there was a total absence of the synthesis of

igninolytic enzymes. These were probably inhibited by the highnitial phenolic content (8.4 g kg−1). Besides, the pasty structuref the medium would not allow a good transfer of oxygen.

The effect of mixing the substrate with the sugarcane bagasse

Please cite this article in press as: F. Aloui et al., Decolorization of semisolid olive residues of “alperujo” during the solid state fermen-tation by Phanerochaete chrysosporium, Trametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),doi:10.1016/j.bej.2007.01.005

o a percentage of 25% of the dry matter was studied. CO2 evolu-ion could be used as an indirect measurement of growth phasesf P. chrysosporium grown on AL. The use of sugarcane bagassemproved the growth, as well as the decolorization of the sub-

Fig. 1. Total amount of CO2 produced per gram dry matter by A. niger (�), P.chrysosporium (�), T. versicolor (�) and P. cinnabarinus (�) grown on sterilizedAL imbibed with basal medium in the absence of a solid support (a), or mixedwith sugarcane bagasse (b).

able 2arameters of fermentation during growth of T. versicolor, P. cinnabarinus, P. chrysosporium and A. niger on AL in absence of a solid support (initial conditions:Hi = 4.82; DMi = 47.2%; COD = 112 g kg−1, phenolic content = 8.4 g kg−1; air = 50 ml min−1, and incubation time = 120 h)

train T. versicolor P. cinnabarinus A. niger P. chrysosporium

inal DM (%) 46.6 47.5 47.1 47.3W (%) 0.3 3.7 1.4 4.8inal pH 4.75 4.39 4.78 4.78inal COD (g kg−1) 112 95.4 101.0 88.7emoval COD (%) 0 14.8 9.8 20.8inal phenolic content (g kg−1) 8.4 7.8 8.4 7.4emoval phenolic content (%) 0 7.1 0 11.9

ARTICLE IN PRESS+ModelBEJ-4418; No. of Pages 6

4 F. Aloui et al. / Biochemical Engineering Journal xxx (2007) xxx–xxx

Table 3Parameters of fermentation and enzymes activities during growth of T. versicolor, P. cinnabarinus, P. chrysosporium and A. niger on AL in presence of sugarcanebagasse as a solid support

Strain T. versicolor P. cinnabarinus A. niger P. chrysosporium

Final DM (%) 37.7 39.0 40.2 33.8LW (%) 11.4 10.8 7.6 19.4Final COD (g kg−1) 54.5 53.4 67.4 52.0Removal COD (%) 40.8 42.0 26.7 43.5Final phenolic content (g kg−1) 3.2 4.3 6.1 2.6Removal phenolic content (%) 47.5 29.5 0 57.4Dec (%) 39.2 21.9 0 39.5LiP (U g−1) 280 0 – 250MnP (U g−1) 610 10 – 160L −1 20

I t = 6.1

scAb(

Iowca

Fpm(

rcafiprt

accases (U g ) 220

nitial conditions: pHi = 4.75; DMi = 39.4%; COD = 92 g kg−1; phenolic conten

trate (Table 3). A reduction of the lag phase to 4 h with P.innabarinus and T. versicolor was observed. On the other hand,. niger growth was inhibited by the substrate as demonstratedy low CO2 production (Fig. 1a) and a reduced COD removalTable 3).

CO2 evolution profile shows the metabolic activity of fungi.n presence of sugarcane bagasse as support, three growth phasesf P. chrysosporium can be clearly observed (Fig. 2). The first

Please cite this article in press as: F. Aloui et al., Decolorization of setation by Phanerochaete chrysosporium, Trametes versicolor, Pycnopodoi:10.1016/j.bej.2007.01.005

as a lag phase (14 h). The second was an accelerated phaseorresponding to the easy substrate consumption (reducing sug-rs and the glycerol). In the third phase, the specific growth

ig. 2. Effect of sugarcane bagasse proportion on the evolution of respirometricarameters of P. chrysosporium grown on sterilized AL imbibed with basaledium at 37 ◦C in SSF: 0% (♦), 10% (�), 20% (�), 30% (�) and 40% (�).

a) Total amount of CO2 produced per gram dry matter and (b) CO2 rate.

bbCcnvtoswAoianetpoE(scmM

tSzcywm

– 0

g kg−1; air = 50 ml min−1, and incubation time = 120 h.

ate was much lower than that of the second phase. This phaseorresponds to the growth on polyphenols. Mixing AL with sug-rcane bagasse clearly increased the specific growth rate of theour strains (Fig. 1b). The transfer of oxygen was significantlymproved by the utilization of the sugarcane bagasse as a sup-ort. In addition, the toxicity was diluted in the medium. Theespiratory quotient (RQ) remained around 1 during the fermen-ation process. This value indicates that CO2 generation wasasically biotic with enough aeration to achieve a complete aero-ic metabolism. Although P. chrysosporium showed the highestOD removal (43.5%) and decolorization activity (39.5% ofolour removal after 5 days followed by the synthesis of ligni-olytic enzymes, LiP = 280 U g−1 and MnP = 160 U g−1), T.ersicolor and P. cinnabarinus also showed a good level ofreatment. However A. niger was able to grow and decrease therganic charge but did not affect the phenolic content. T. ver-icolor was found to efficiently decolorize the AL. This strain,hich produced LiP, MnP and laccases enzymes, decolorized theL to a lesser extent than P. chrysosporium, which expressednly LiP and MnP. Moreover, P. cinnabarinus, which exhib-ted only MnP and laccases, also decolorized AL. Hence, thebility of T. versicolor to produce laccases (phenol oxidase) didot enhance the decolorization of AL. This phenomenon can bexplained by the fact that in a system where the LiP is present,he laccase oxidizes the simple phenolic compounds, causingolymerisation reactions. This was not the case when there werenly laccase and MnP enzymes. This is in line with Sayadi andllouz. [21], who showed that the WRF Dichomitus squalens

MnP, laccases) degrade the lignin more efficiently than the twotrains: Coriolus versicolor and Phlebia radiata (LiP, MnP, lac-ases). Boer et al. [22] showed that MnP appeared to be theain responsible for the capability of Lentinula edodes (LiP,nP, laccases) to decolorize synthetic dyes.Decolorization of AL in submerged fermentation was lower

han that in SSF. This can be explained by the fact that inSF, enzymes secreted by mushrooms tend to remain in a nearone of the organism and are not immediately diluted as in the

misolid olive residues of “alperujo” during the solid state fermen-rus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),

ase of the liquid cultures. This reduced diffusion rate wouldield more efficient enzymes. For this type of fermentation, theater activity is very low. Therefore, the produced enzymes areore concentrated in the medium and should be more efficient.

IN PRESS+ModelB

ineering Journal xxx (2007) xxx–xxx 5

Bmessw

sgwsafdttd

dbrt

3t

ci

adstpacocpprevonsttwioytAoa

Fig. 3. Effect of sugarcane bagasse proportion on the evolution of lignin peroxi-dase (�), manganese peroxidase (�) and decolorization (�) by P. chrysosporiumgrown on sterilized AL imbibed with basal medium at 37 ◦C in SSF.

Table 4Effect of sugarcane bagasse proportion on the evolution of COD and phenoliccontent by P. chrysosporium grown on sterilized AL imbibed with basal mediumat 37 ◦C in SSF

Sugarcane bagasse proportion 0% 10% 20% 30% 40%

Initial COD (g kg−1) 109.4 103.6 97.8 92.4 85.3Final COD (g kg−1) 87.7 72.7 56.2 45.2 45.4Removal COD (%) 19.8 29.8 42.5 51.1 46.8Initial phenolic content (g kg−1) 8.7 8.2 7.1 6.4 5.9FR

fanmtiaaohtft

dratfaui

4

ARTICLEEJ-4418; No. of Pages 6

F. Aloui et al. / Biochemical Eng

esides, in SSF, the transfer of oxygen is higher than in sub-erged fermentation. Consequently, a high level of ligninolytic

nzymes could be produced and the decolorization of the sub-trate could be enhanced. This finding confirms previous resultstating that the activities of the enzymes LiP and MnP in SSFere far higher than in submerged fermentation [23].Cultivation of the four strains on AL in absence of a solid

upport was not well established, because the AL has not aood structure for oxygen transfer, also the growth of myceliaas very poor. However, P. chrysosporium and P. cinnabarinus

howed a slight growth. The use of the sugarcane bagasse assolid support mixed with the substrate, improved gas trans-

er and the growth of the four strains, but the best results ofecolorization and exhaustion of the COD were obtained withhe three WRF. Basidiomycetes are known by their capabilityo mineralise the lignin efficiently and therefore can be used toepolymerise the existing polyphenols in the substrate.

The results obtained show that the ligninolytic complex pro-uced by P. chrysosporium cultures was able to decolorize ALetter than the other fungi. This is in agreement with the resultseported in previous works [24,25]. This strain needs to be fur-her studied to improve the treatment of AL in SSF.

.2. Effect of sugarcane bagasse proportion on thereatment of AL substrate by P. chrysosporium in SSF

In order to improve the treatment of AL substrate by P.hrysosporium, sugarcane bagasse proportions (0–40%, w/w)n the medium were optimised.

The results obtained confirmed the importance of the sug-rcane bagasse as a support. Numerous SSF processes wereeveloped in which sugarcane bagasse was used as the solid inertupport [26]. A high proportion of sugarcane bagasse improvedhe growth determined by respirometry, while increasing theroduction of CO2 (Fig. 2a) and decreasing the lag phase. Thenalysis of CO2 concentrations for every proportion of sugar-ane bagasse (Fig. 2b) showed an optimum support proportionf 30%. Indeed, when comparing the first peak of CO2 (Fig. 2b)orresponding to every experiment, it can be noticed that thiseak was more important at 30% of sugarcane bagasse. The firsthase represents the vegetative phase of P. chrysosporium. It cor-esponds to the growth on the easier substrates (reducing sugarsxisting in the AL). The peaks of CO2 rate obtained after theegetative phase correspond to the growth of P. chrysosporiumn the recalcitrant substrates in the medium (especially polyphe-ols). Fig. 2b shows the importance of these peaks at 30% ofugarcane bagasse. The physico-chemical analyses confirmedhe results of the respirometry. Indeed, the optimum decoloriza-ion, as well as the maximal activities of ligninolytic enzymesas obtained at 30% of sugarcane bagasse (Fig. 3). Also max-

mum removal COD and phenolic content were obtain at 30%f sugarcane bagasse (Table 4). For this case, BOD was anal-sed, and the ration COD/BOD of treated AL was estimated

Please cite this article in press as: F. Aloui et al., Decolorization of setation by Phanerochaete chrysosporium, Trametes versicolor, Pycnopodoi:10.1016/j.bej.2007.01.005

o 3.8 demonstrating the stabilized state of the organic matterccording to these results, one should note the positive effectf sugarcane bagasse used as a support to improve the growthnd the decolorization while increasing the gas exchange sur-

ci

inal phenolic content (g kg−1) 7.6 5.5 3.2 2.5 2.8emoval phenolic content (%) 12.6 32.9 54.9 60.9 52.5

ace. Indeed, in the absence of the sugarcane bagasse, there wastotal absence of both decolorization, and the synthesis of ligni-olytic enzymes. The ligninolytic system of P. chrysosporiumust be totally inhibited. As was explained in previous works,

he sugarcane bagasse improves the diffusion of the oxygen thats a main factor of the decolorization [27]. As a consequence,free circulation of air requires a sufficiently porous medium

s was recommended by Raimbault [9]. The fibrous structuref sugarcane bagasse mixed to AL substrate seems to allow theumidified air to cross the mass of the product easily all alonghe incubation period. This would provide the necessary oxygenor the growth of the strain, without avoiding the desiccation ofhe product.

AL decolorization can prove a toxicity decrease. In fact, theegradation of phenolic substrate like AL or OMW by Phane-ochaete sp., has been investigated by several researches groupnd it has been demonstrated that phenolic compounds concen-ration and OMW toxicity decrease whereas the color changerom black to yellow [28,29]. This is online with Dhouib etl. [30], who show that compared to the 100% inhibition ofntreated OMW, P. chrysosporium decreased the relative toxic-ty in a range of 70% using the Vibrio fisheri luminescence.

. Conclusion

misolid olive residues of “alperujo” during the solid state fermen-rus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),

It can be concluded that among the different strains tested, P.hrysosporium seemed to be the best fungus to treat AL substraten SSF. On the other hand, extracellular fluid (composed mainly

IN+ModelB

6 ineer

oe

cs

cw

A

aTtS

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ARTICLEEJ-4418; No. of Pages 6

F. Aloui et al. / Biochemical Eng

f LiP) exhibited a high ability to decolorize AL showing thefficiency of the enzyme produced.

These promising results suggest the application of P.hrysosporium to large-scale processes in order to treat ALubstrate in SSF.

Further work is needed to establish whether the treated ALould be further processed into feed for animals such as poultryithout presenting any risk.

cknowledgments

This work was performed under research and cooperationgreements between the CBS (Tunisia) and the IRD (France).he authors would like to thank M. Raimbault for his help in

he realisation of this work, and A. Hajji from the Engineeringchool of Sfax for his help with English.

eferences

[1] M. Hamdi, A. Kadir, J.L. Garcia, The use of Aspergillus niger for biocon-version of olive mill waste-waters, Appl. Microbiol. Biotechnol. 34 (1991)829–831.

[2] B.W. Bogan, R. Lamar, One-electron oxidation in the degradation of cre-osote polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium,Appl. Environ. Microbiol. 61 (1995) 2631–2635.

[3] C. Laugero, C. Mougin, J.C. Sigoillot, S. Moukha, M. Asther, Comparisonof static and agitated immobilized cultures of Phanerochaete chrysospo-rium for the degradation for the degradation of pentachlorofenol and itsmetabolite pentachloroanisole, Can. J. Microbiol. 43 (1997) 378–383.

[4] J.A. Alburquerque, J. Gonzalvez, D. Garc�a, J. Cegarra, Agrochemicalcharacterisation of “alperujo”, a solid by-product of the two-phase centrifu-gation method for oil extraction, Bioresour. Technol. 91 (2004) 195–200.

[5] B.K. Lonsane, N.P. Ghildyal, S. Budiatman, S.V. Ramakrishna, Engineer-ing aspects of solid state fermentation, Enzyme Microbiol. Technol. 7(1985) 258–265.

[6] G. Gutierrez-Sanchez, S. Roussos, C. Augur, Effect of the nitrogen sourceon caffeine degradation by Aspergillus tamarii, Lett. Appl. Microbiol. 38(2003) 50–55.

[7] M.V. Ramesh, B.K. Lonsane, Ability of solid state fermentation techniqueto significantly minimize catabolic repression of alpha-amylase productionby Bacillus licheniformis M27, Appl. Microbiol. Biotechnol. 35 (1991)591–593.

[8] N. Roche, A. Venague, C. Desgranges, A. Durand, Use of chitin measure-ment to estimate fungal biomass in solid state fermentation, Biotechnol.Adv. 11 (1993) 677–683.

[9] M. Raimbault, Ph.D. Thesis, Universite Paul Sabatier de Toulouse, France,1980.

10] G. Saucedo-Castaneda, M.R. Trejo-Hernandez, B.K. Lonsane, J.M.Navarro, S. Roussos, D. Dufour, M. Raimbault, On line automated monitor-ing and control systems for CO2 and O2 in aerobic and anaerobic solid-statefermentations, Process Biochem. 29 (1994) 13–24.

Please cite this article in press as: F. Aloui et al., Decolorization of setation by Phanerochaete chrysosporium, Trametes versicolor, Pycnopodoi:10.1016/j.bej.2007.01.005

11] M. Raimbault, D. Alazard, Culture method to study fungal growth in solidfermentation, Eur. J. Appl. Microbiol. Biotechnol. 9 (1980) 199–209.

12] J.D. Box, Investigation of the Folin-Ciocalteau phenol reagent for the deter-mination of polyphenolic substances in natural waters, Water Res. 17 (1983)511–522.

[

PRESSing Journal xxx (2007) xxx–xxx

13] R.J. Knechtel, A more economical method for the determination of chem-ical oxygen demand, Water Pollut. Control (1978) 25–29.

14] S. Sayadi, N. Allouche, M. Jaoua, F. Aloui, Detrimental effects of highmolecular-mass polyphenols on olive mill wastewater biotreatment, Pro-cess Biochem. 35 (2000) 725–735.

15] M. Tien, T.K. Kirk, Lignin-degrading enzyme from Phanerochaetechrysosporium: purification, characterization and catalytic cycle of aunique H2O2-requiring oxygenase, Proc. Natl. Acad. Sci. U.S.A. 81 (1984)2280–2284.

16] A. Paszcynski, V.B. Huynh, R. Crawford, Enzymatic activities ofan extracellular manganese dependent peroxidase from Phanerochaetechrysosporium, FEMS Microbiol. Lett. 29 (1985) 37–41.

17] G. Szklarz, R. Antibus, R. Sinsabaugh, A. Linkinns, Production of phenoloxydases and peroxidases by wood rotting fungi, Mycologia 81 (1989)234–240.

18] J. Pintado, B.K. Lonsane, I. Gaime-Perraud, S. Roussos, On line monitoringof citric acid production in solid-state culture by respirometry, ProcessBiochem. 33 (5) (1998) 513–518.

19] S. Roussos, M. Raimbault, Hydrolyse de la cellulose par les moisissures.1. “Screening” des souches cellulolytiques, Ann. Microbiol. Inst. Pasteur.133 (1982) 455–464.

20] F. Priego-Capote, J. Ruiz-Jimenez, M.D. Luque de Castro, Fast separationand determination of phenolic compounds by capillary electrophoresis-diode array detection: application to the characterisation of alperujoafter ultrasound-assisted extraction, J. Chromatogr. A 1045 (2004)239–246.

21] S. Sayadi, R. Ellouz, Screening of white rot fungi for the treatment of olivemill waste-waters, J. Chem. Technol. Biotechnol. 57 (1993) 141–146.

22] C.G. Boer, L. Obici, C.G. Marques de Souza, R.M. Peralta, Decolorizationof synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes pro-ducing manganese peroxidase as the main ligninolytic enzyme, Bioresour.Technol. 94 (2004) 107–112.

23] X. Fujian, C. Hongzhang, L. Zuohu, Solid-state production of ligninperoxidase (LiP) and manganese peroxidase (MnP) by Phanerochaetechrysosporium using steam-exploded straw as substrate, Bioresour. Tech-nol. 80 (2001) 149–151.

24] S. Sayadi, R. Ellouz, Decolorization of olive mill waste-waters by Phane-rochaete chrysosporium: involvement of the lignin degrading system, Appl.Microbiol. Biotechnol. 37 (1992) 813–817.

25] S. Sayadi, F. Zorgani, R. Ellouz, Decolorization of olive mill waste-watersby free and immobilized Phanerochaete chrysosporium, Appl. Biochem.Biotechnol. 56 (1996) 265–276.

26] A. Pandey, C.R. Soccol, P. Nigam, V.T. Soccol, Biotechnological potentialof agro-industrial residues. I. Sugarcane bagasse, Bioresour. Technol. 74(2000) 69–80.

27] R.E. Mudget, A.J. Paradis, Solid state fermentation of natural birch ligninby Phanerochaete chrysosporium, Enzyme Microbiol. Technol. 7 (1985)150–154.

28] A. Tsioulpas, D. Dimou, D. Iconoumou, G. Aggelis, Phenolic removal inolive oil mill wastewater by strains of Pleurotus sp. In respect to theirphenoloxidase (laccase) activity, Bioresour. Technol. 84 (2002) 251–257.

29] G. Aggelis, D. Iconoumou, M. Christou, D. Bokas, S. Kotzailias, G. Chris-tou, V. Tsagou, S. Papanikolaou, Phenolic removal in a model olive oil millwastewater using Pleurotus ostreatus in bioreactor cultures and biological

misolid olive residues of “alperujo” during the solid state fermen-rus cinnabarinus and Aspergillus niger, Biochem. Eng. J. (2007),

evaluation of the process, Water Res. 37 (2003) 3897–3904.30] A. Dhouib, F. Aloui, N. Hamad, S. Sayadi, Complete detoxification of olive

mill wastewaters by integrated treatment using the white rot fungus Phane-rochaete chrysosporium followed by anaerobic digestion and ultrafiltration,Biotechnology 4 (2) (2005) 153–162.