Chemosphere 88 (2012) 620–626

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Chemosphere

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Biodegradation and detoxification of olive mill wastewater by selected strainsof the mushroom genera Ganoderma and Pleurotus

Spyridon Ntougias a,d, Petr Baldrian b, Constantinos Ehaliotis c, Frantisek Nerud b, Theodoros Antoniou d,Vera Merhautová b, Georgios I. Zervakis d,e,⇑a Democritus University of Thrace, Department of Environmental Engineering, Laboratory of Wastewater Management and Treatment Technologies, Vas. Sofias 12, 67100Xanthi, Greeceb Institute of Microbiology of the ASCR, Videnska 1083, 14220 Prague, Czech Republicc Agricultural University of Athens, Laboratory of Soils and Agricultural Chemistry, Iera Odos 75, 11855 Athens, Greeced National Agricultural Research Foundation, Institute of Kalamata, Lakonikis 87, 24100 Kalamata, Greecee Agricultural University of Athens, Department of Agricultural Biotechnology, Laboratory of General and Agricultural Microbiology, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 November 2011Received in revised form 28 February 2012Accepted 9 March 2012Available online 3 April 2012

Keywords:White-rot fungiMushroomToxicityOlive-oil mill phenolicsOMW decolorization

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.03.042

⇑ Corresponding author at: Agricultural UniversitAgricultural Biotechnology, Laboratory of General anIera Odos 75, 11855 Athens, Greece. Tel.: +30 210529

E-mail address: zervakis@aua.gr (G.I. Zervakis).

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initiallyscreened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganodermaaustrale, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further exam-ined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cul-tures resulted in significant decolorization (by 40–46% and 60–65% for Ganoderma and Pleurotus spp.respectively) and reduction of phenolics (by 64–67% and 74–81% for Ganoderma and Pleurotus spp.respectively). COD decrease was less pronounced (12–29%). Cress-seeds germination increased by 30–40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeriluminescence was reduced in fungal-treated OMW samples by approximately 5–15 times compared tothe control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase weredetected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhi-bit any enzyme activities at early growth stages; instead, high laccase (five times greater than those ofGanoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolor-ization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii lac-case activity was correlated with the effluent’s decolorization.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Olive mill wastewater (OMW) is among the most notorioussources of environmental pollution in olive-oil producing coun-tries, mainly because of its high content in polyphenolics and totalorganic matter; furthermore, the seasonal operation and the widegeographic distribution of three-phase olive mills create additionaldifficulties for its effective management (Ouzounidou et al., 2010).

White-rot basidiomycetes are among the most potent organ-isms to biodegrade and detoxify a wide range of wastes and pollu-tants. These fungi selectively attack lignin and related compoundsby producing one or more of phenol-targeting redox enzymes,namely the peroxidases and laccases/phenoloxidases (Martinezet al., 2005; Baldrian, 2006). Since many of the organic compounds

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y of Athens, Department ofd Agricultural Microbiology,4341; fax: +30 2105294344.

present in OMW are identical or very similar to lignin biodegrada-tion products, ligninolytic fungi have been exploited for the biore-mediation of OMW through dephenolization, COD reduction and/or decolorization (D’Annibale et al., 2004; Dias et al., 2004; Ergület al., 2009). Pleurotus species are among those organisms demon-strating the potential for biodegrading the effluent (Tsioulpas et al.,2002), which could be combined with the generation of value-added products, e.g. edible mushrooms (Zervakis et al., 1996). Onthe other hand, the use of Ganoderma (i.e., G. applanatum) forOMW treatment was only scarcely reported (Matos et al., 2007).

In the present study, a screening procedure was adopted to se-lect the most effective OMW degraders among 39 white-rot fungibased on their ability to reduce the phenolic content of OMW.Activities of ligninolytic/phenol-degrading enzymes produced byselected Pleurotus and Ganoderma strains were examined in respectto OMW decolorization, reduction of phenolics content, COD de-crease and toxicity for evaluating the effluent’s biodegradationprocess.

S. Ntougias et al. / Chemosphere 88 (2012) 620–626 621

2. Materials and methods

2.1. Fungal strains

Thirty-nine white-rot fungal strains belonging to nine species ofthe subphylum Agaricomycotina (Basidiomycota) (Table 1), wereevaluated for their potential to decrease the OMW phenoliccontent.

2.2. Physicochemical characteristics of olive mill wastewater

Olive mill wastewater (OMW) was obtained from an olive oilmill equipped with three-phase centrifugal decanters located inKalamata (SW Greece). The main parameters characterizing thiseffluent were: Total organic carbon (TOC): 23 g L�1, total N:0.61 g L�1, P2O5: 0.26 g L�1, K2O: 1.98 g L�1, total phenolics:4.9 mg mL�1, chemical oxygen demand (COD): 47 g L�1, total sus-pended solids (TSSs): 37 g L�1 (Ntougias et al., 2010).

Measurements of pH and electrical conductivity (EC; i.e., esti-mate of the total amount of ions in solution) in OMW wereperformed using a Scott Geräte TR156 pH-meter and a Wissenchaft-lich-Technische Werkstätten LF530 conductivity meter respec-tively. Cu, Fe, Mn and Zn were determined by using flame atomicabsorption spectrophotometry (AAS, Aurora Instruments, Canada)in triplicate samples as described previously (Baldrian et al., 2005).

Table 1Effect of 39 white-rot fungi growing in 25% v/v OMW on the reduction of total phenolics dOne-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05) was used to evindicates statistically significant differences (Tukey’s test, p < 0.05). NA: not applicable.

a/a Fungal strain

1 Pleurotus ostreatus LGMACC 222 Ganoderma australe IK-16003 Ganoderma carnosum IK-16424 Pleurotus eryngii LGAM P665 Pleurotus ostreatus LGAM P686 Pleurotus pulmonarius LGAM P107 Pleurotus pulmonarius LGAM P168 Pleurotus eryngii CBS 100829 Pleurotus pulmonarius LGAM P3

10 Pleurotus ostreatus LGAM P7211 Pleurotus eryngii LGAM P6312 Pleurotus pulmonarius LGAM P4213 Pleurotus eryngii LGMACC 85110114 Pleurotus pulmonarius LGAM P4615 Pleurotus eryngii D-183216 Pleurotus pulmonarius LGAM P717 Pleurotus eryngii UPA1218 Pleurotus eryngii LGAM P10119 Lentinula edodes SIEF023120 Lentinula edodes SIEF023221 Auricularia auricula-judae SIEF008922 Pleurotus eryngii UPA3023 Pleurotus ostreatus LGMACC 85040124 Pleurotus cornucopiae ATCC 3854725 Pleurotus eryngii UPA526 Pleurotus eryngii LGAM P6527 Pleurotus nebrodensis UPA2828 Lentinula edodes IK-1229 Pleurotus nebrodensis UPA830 Pleurotus eryngii UPA3131 Pleurotus eryngii LGAM P10232 Pleurotus eryngii UPA1033 Pleurotus pulmonarius LGAM P1134 Pleurotus nebrodensis UPA735 Pleurotus pulmonarius LGAM P3736 Pleurotus eryngii LGAM P10937 Pleurotus eryngii LGMACC 83110138 Pleurotus ostreatus LGAM P6139 Pleurotus ostreatus LGAM P3840 Control (25% v/v OMW)

2.3. Growth media and conditions

The growth medium consisted of OMW diluted in water at a 1:3volume ratio (25% v/v OMW), adjusted to pH 6 by the addition ofCaO, centrifuged for 5 min at 4250 g, and heat-sterilized for20 min (121 �C, 1.1 atm). Fungal strains were maintained on 25%v/v OMW medium solidified with 1.7% w/v agar. Agar plugs(6 mm diameter), originating from the actively growing part ofthe mycelium developing on solidified OMW, were used to inocu-late static liquid cultures (100 ml of 25% v/v OMW).

Fungal strains were evaluated by measuring the residual pheno-lic content in liquid cultures after an incubation period of 35 d at25 �C. At a second stage, the four most effective fungal strains in re-spect to total phenolics reduction, were further evaluated; thistime, measurements were performed 0, 10, 20 and 30 d after inoc-ulation of liquid OMW media. Four replicates were tested for eachfungal strain studied. In all experiments performed, non-inocu-lated liquid media (25% v/v OMW) were used as controls.

2.4. Determination of fungal biomass, OMW decolorization, COD, totalphenolics and germination indices

Biomass was harvested by filtration, and mycelium dry weightwas determined by drying at 70 �C until constant weight.

Decolorization was estimated by measuring the absorbance ofinoculated and non-inoculated OMW samples at 525 nm

uring a 35-d incubation period. Values are expressed as mean ± standard error (n = 3).aluate statistical differences among the values obtained. Lack of letters in common

Total phenolics (g L�1) Phenolics content reduction (%)

0.26 ± 0.05 (a) 78.00.29 ± 0.02 (ab) 75.40.29 ± 0.01 (ab) 75.40.34 ± 0.03 (ab) 71.20.35 ± 0.02 (a–c) 70.30.35 ± 0.04 (a–d) 70.30.37 ± 0.10 (a–d) 68.60.36 ± 0.07 (a–d) 69.50.38 ± 0.09 (a–d) 67.80.40 ± 0.02 (a–e) 66.10.40 ± 0.05 (a–e) 66.10.42 ± 0.03 (a–e) 64.40.42 ± 0.07 (a–e) 64.40.43 ± 0.07 (a–e) 63.60.52 ± 0.01 (a–f) 55.90.53 ± 0.10 (a–g) 55.10.58 ± 0.05 (a–h) 50.80.63 ± 0.05 (a–i) 46.60.67 ± 0.02 (b–j) 43.20.74 ± 0.02 (c–k) 37.30.75 ± 0.17 (d–k) 36.40.79 ± 0.09 (e–l) 33.10.83 ± 0.22 (f–l) 29.70.88 ± 0.06 (f–l) 25.40.90 ± 0.02 (f–l) 23.70.93 ± 0.02 (g–l) 21.20.94 ± 0.01 (h–l) 20.30.94 ± 0.06 (h–l) 20.30.96 ± 0.05 (h–l) 18.60.97 ± 0.06 (h–l) 17.80.99 ± 0.04 (i–l) 16.11.02 ± 0.03 (i–l) 13.61.04 ± 0.03 (j–l) 11.91.05 ± 0.06 (j–l) 11.01.05 ± 0.04 (j–l) 11.01.06 ± 0.10 (j–l) 10.21.07 ± 0.07 (k–l) 9.31.15 ± 0.11 (l) 2.51.16 ± 0.04 (l) 1.71.18 ± 0.01 (l) NA

622 S. Ntougias et al. / Chemosphere 88 (2012) 620–626

(Aggelis et al., 2002) using a U-2001 spectophotometer(Hitachi Instruments Inc., USA).

Dissolved chemical oxygen demand (COD) was determinedusing the Standard Methods for the Examination of Water andWastewater (Clesceri et al., 1998).

Total phenolics were analyzed by the Folin–Ciocalteu method(Singleton and Rossi, 1965).

The seed germination index (GI) was estimated according toZucconi et al. (1981) protocol by using 25 cress (Lepidium sativumL.) seeds per sample; these were placed onto a filter paper moist-ened either with the sample or with water (control), and they wereincubated in a Petri dish for 3 d in the dark. GI was calculatedaccording to the following formula:

GIð%Þ

¼ number of viable seeds in the sample x root length of the seeds in the samplenumber of viable seeds in the control x root length of the seeds in the control

�100%

2.5. Toxicity test

Toxicity was measured for treated samples and control after 30-d incubation by monitoring the inhibition of the luminescence ofAliivibrio fischeri (ex. Vibrio fischeri) according to the German stan-dard procedure, DIN 38412 L341 1994.

The loss of luminescence due to absorption by colored OMWsamples was corrected by measuring several dilutions in a cuvetteluminometer (LumisTox; Dr. Lange GmbH, Germany) using the col-or-correction method described by Klein (1990).

2.6. Determination of enzymes activities

Laccase (Lac, EC 1.10.3.2: benzenediol: oxygen oxidoreductase)activity was determined spectrophotometrically by monitoring theoxidation of ABTS (2,20-azinobis-3-ethylbenzothiazoline-6-sulfonicacid) in citrate–phosphate (100 mM citrate, 200 mM phosphate)buffer (pH 5.0) at 420 nm (Bourbonnais and Paice, 1990).

Manganese-independent peroxidase (MnIP) and manganeseperoxidase (MnP, EC 1.11.1.13 (MnII): hydrogen-peroxide oxidore-ductase) activities were determined in succinate-lactate buffer(100 mM, pH 4.5) as described by Aggelis et al. (2002) and Ngoand Lenhoff (1980) respectively. MBTH (3-methyl-2-benzothiazoli-none hydrazone) and DMAB (3,3-dimethylaminobenzoic acid)served as substrates and were oxidatively coupled by the enzymes,and the resulting purple indamine dye was detected at 595 nm.

Lignin peroxidase (LiP, E.C.1.11.1.14: diarylpropane: oxygen,hydrogen-peroxide oxidoreductase) activity was determined byusing veratryl alcohol (20 mM) as substrate in the presence ofH2O2 (54 mM) in Na-tartrate buffer (0.1 M, pH 3) at 310 nm (Tienand Kirk, 1984). Veratryl alcohol oxidase activity (VAOx) was esti-mated following the protocol for LiP in the absence of H2O2.

For all enzyme determinations, one activity unit was defined asthe amount of enzyme transforming 1 lmol of substrate perminute.

2.7. Statistical analysis

Analysis of Variance (ANOVA) was performed, followed byTukey’s multiple comparison tests, to evaluate statistical signifi-cance of differences between treatment means. Standard errorswere calculated for all mean values and regression analysis wascarried out to assess relationships between variables. Differencesat p < 0.05 were considered statistically significant.

3. Results

The contents of Cu, Fe, Mn and Zn in the initial OMW (100% v/v)were 0.15 ± 0.02, 2.31 ± 0.03, 0.49 ± 0.02 and 0.86 ± 0.01 mg L�1

respectively, indicating sufficient concentrations of metals re-quired by phenol-oxidizing oxidoreductases in 25% v/v OMW sam-ples. The comparative evaluation of 39 fungal strains revealed thattheir ability to reduce OMW phenolics content appears to bestrain- and not species-dependent, since high pertinent intraspe-cific variability was observed (Table 1). Hence, four white-rot fungiwere selected as the most efficient OMW-phenolics degraders(phenolic content reduced by 71–78%): Pleurotus ostreatusLGMACC 22, Ganoderma australe IK-1600, Ganoderma carnosumIK-1642 and Pleurotus eryngii LGAM P66.

The selected strains grew well in static batch cultures; biomassproduction for Ganoderma strains was significantly higher (by atleast 27%) when compared to the biomass of Pleurotus strains after20 d of incubation. Similarly, significant higher biomass productionwas observed for G. australe IK-1600 as compared to the biomassproduced by G. carnosum IK-1642 and P. ostreatus LGMACC 22 atthe end of the 30-d incubation period (Table 2).

P. ostreatus and P. eryngii strains demonstrated high decoloriza-tion efficacy on the OMW medium, i.e., by 41.4–44.0% and60.4–64.8% at the 20-d and 30-d incubation periods respectively;no color reduction was detected after 10 d of incubation (Fig. 1).G. australe and G. carnosum treatments initially presented an in-creased intensity in OMW color, which was however followed bysignificant net decolorization (40.2% and 31.5%) after a 20-d incu-bation period (Fig. 1).

The phenolic content was considerably reduced by Ganodermastrains, i.e., by 40.8–43.5% and 60.4–65.0% (compared to OMWcontrol) at the end of the 10-d and 20-d incubation periods respec-tively (Fig. 2). In contrast, no phenolics reduction was initially de-tected for Pleurotus strains; however, a significant pertinentdecrease by 62.5% and 73.9% was observed after 20 d of incubationfor P. ostreatus and P. eryngii respectively, followed by a furtherslight reduction during the last 10 d of incubation.

As regards COD values, the greatest decrease (by 28.5%) wasdemonstrated by G. carnosum, while the other three strains re-duced COD by 12.0–18.7% (Table 3). Values for pH remainedslightly acidic in Ganoderma strains growth media (e.g. pH: 6.00for G. carnosum), whereas they increased in Pleurotus substrates(7.42–8.31) (Table 3). Moreover, Pleurotus spp. showed higherreduction (by 19.4–25.8%) of the OMW medium’s electrical con-ductivity compared to Ganoderma strains (Table 3).

Plant-seed germination indices did not alter significantly duringthe first 10 d of incubation for all strains; from then on, OMWtreatment with Pleurotus strains revealed decreased phytotoxicity,especially in the case of P. eryngii which presented a germinationincrease by 26.9 percentage units after 20-d incubation period(Fig. 3). P. ostreatus was also efficient in reducing phytotoxicity(by 18.9 percentage units in respect to OMW control) after 30 dof incubation (Fig. 3). In contrast, OMW treatment by G. carnosumdid not substantially affect germination indices, while an increasein phytotoxicity was observed for G. australe at the end of cultiva-tion (Fig. 3).

As regards the evaluation of OMW toxicity through the lumi-nescence inhibition of A. fischeri, all treatments revealed signifi-cantly lower toxicity values compared to the control (Table 3).The highest decrease (by ca. 15 times) of toxicity was detected inOMW treated by P. eryngii; Ganoderma strains presented relativelyhigh reduction of toxicity as well (i.e., by 5–8 times as compared tothe control).

Laccase activities presented a specific pattern for each one ofthe two genera examined. Hence, Ganoderma strains showed Lac

Table 2Laccase, manganese peroxidase and manganese-independent peroxidase activities exhibited by selected fungal strains during a 30-d incubation period. Biomass productionvalues (expressed in grams – d.w. – of harvested mycelium from 100 ml of 25% v/v OMW) are presented at the bottom of the Table. Enzyme activities and biomass productionvalues are expressed as mean ± standard errors (n = 4). Lack of letters in common indicates statistically significant differences (Tukey’s test, p < 0.05) between treatment means atdifferent time-periods for each strain (capital letters), and at the same time period among the four fungal strains (lowercase letters).

Enzymatic activity (U L�1) Ganoderma australe IK-1600 Ganoderma carnosum IK-1642 Pleurotus ostreatus LGMACC 22 Pleurotus eryngii LGAM P66

LaccaseDay 10 8.34 ± 1.88 (b, A) 10.04 ± 0.85 (b, A) 0 (a, A) 0 (a, A)Day 20 18.47 ± 1.32 (a, B) 21.30 ± 0.81 (a, B) 57.35 ± 12.38 (b, B) 73.95 ± 4.04 (b, B)Day 30 5.11 ± 0.81 (a, A) 10.37 ± 0.79 (a, A) 117.82 ± 10.44 (b, C) 106.85 ± 4.32 (b, C)

Manganese peroxidaseDay 10 0 (A) 0 (A) 0 (A) 0 (A)Day 20 4.29 ± 1.04 (b, B) 0 (a, A) 0 (a, A) 4.52 ± 1.06 (b, B)Day 30 2.52 ± 0.85 (a, AB) 1.04 ± 0.66 (a, A) 29.76 ± 4.68 (b, B) 5.48 ± 1.22 (a, B)

Manganese independent peroxidaseDay 10 30.93 ± 0.82 (b, C) 36.09 ± 2.08 (c, C) 0.74 ± 0.09 (a, A) 2.21 ± 0.66 (a, A)Day 20 12.67 ± 0.38 (a, B) 20.25 ± 4.63 (ab, B) 23.42 ± 1.81 (b, B) 18.56 ± 0.52 (ab, C)Day 30 5.82 ± 0.14 (a, A) 6.11 ± 0.64 (a, A) 25.70 ± 0.39 (c, B) 11.12 ± 1.59 (b, B)

Biomass (g)Day 10 0.005 ± 0.000 (a, A) 0.015 ± 0.002 (b, A) 0.005 ± 0.001 (a, A) 0.004 ± 0.001 (a, A)Day 20 0.117 ± 0.001 (c, B) 0.088 ± 0.005 (b, B) 0.048 ± 0.005 (a, B) 0.064 ± 0.003(a, B)Day 30 0.146 ± 0.003 (b, C) 0.113 ± 0.011 (a, B) 0.115 ± 0.005 (a, C) 0.130 ± 0.006 (ab, C)

ab, A - controlb, A

c, A

bc, C

A for control B for IK-1600C for IK-1642

C for LGMACC 22C for LGAM P66

a, A b, A

c, D

a, B

b, A

ab, C - LGMACC 22

a, B

a, A

a, C

a, B

a, A

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 5 10 15 20 25 30

Time (days)

Abs

orba

nce

(525

nm

)

control

G. australe IK-1600

G. carnosum IK-1642

P. ostreatus LGMACC 22

P. eryngii LGAM P66

Fig. 1. Effect of the growth of four selected fungal strains in 25% v/v OMW on medium’s decolorization during a 30-d incubation period. Error bars represent standard errorsof the means (n = 4). Lack of letters in common indicates statistically significant differences (Tukey’s test, p < 0.05) between treatment means at different time-periods foreach strain (capital letters), and at the same time period among the four fungal strains (lowercase letters).

S. Ntougias et al. / Chemosphere 88 (2012) 620–626 623

activities from the beginning of their incubation, reached a peakduring the second 10-d growth period, and they gradually subsidedtowards the end of the cultivation (Table 2). Both Ganoderma spp.presented similar values of Lac activities (18.5–21.3 U L�1). On theother hand, Pleurotus strains were ‘‘slow starters’’ (zero activity at10-d of incubation); however, during the second and the third per-iod of the 30-d growth, Lac activities increased sharply and finallyreached values of 107 and 118 U L�1 for P. eryngii and P. ostreatusrespectively (Table 2).

No Mn-peroxidase activity was detected at the first 10 d ofincubation for all strains studied; then, at the 20-d period, rela-tively low activities were measured only for G. australe and P.eryngii (4.3 and 4.5 U L�1 respectively) (Table 2). However, atthe end of the incubation, P. ostreatus demonstrated MnP activity(29.8 U L�1) which was significantly higher than the rest of thestrains examined (Table 2). Mn-independent peroxidase activitywas detected at all time-periods for all four strains. Noteworthy

was that the two Ganoderma species presented similar patternsin their respective enzyme activities by demonstrating peak val-ues at 10-d of growth (30.9 and 36.1 U L�1), which declined to-wards the end of incubation (5.8 and 6.1 U L�1) (Table 2). Incontrast, very low MnIP activities were measured for bothPleurotus strains at 10-d of incubation; then P. ostreatus presentedmaximum enzyme production (23.4 and 25.7 U L�1) during thesecond half of the incubation period, whereas P. eryngii producedhighest activities after 20 d of growth (i.e., 18.6 U L�1) (Table 2).No LiP and/or VAOx activity was detected in OMW samples trea-ted by the four selected fungi.

Decolorization of the OMW medium was strongly correlated tothe reduction of phenolics for G. australe and G. carnosum,(R2 = 0.995 and 0.996, respectively). Laccase activity was relatedto the decolorization invoked by P. eryngii (R2 = 0.998), whereasreduction of OMW phenolic content was correlated with the Mn-independent peroxidase (R2 = 0.996) produced by P. ostreatus.

d, Ac, A

b, A -control

bc, A b, A -IK1600

a, B

c, A

b, B - IK1642

a, C

ab, A

b, B -LGMACC 22

b, C - LGMACC 22

a, A

a, B

A for controlC for IK-1600D for IK-1642

C for LGAM P66C for LGAM P66

b, C - LGAM P66

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0 5 10 15 20 25 30Time (days)

Tota

l phe

nolic

s(e

xpre

ssed

as

mg

mL-1

syr

ingi

c ac

id)

controlG. australe IK-1600G. carnosum IK-1642P. ostreatus LGMACC 22 P. eryngii LGAM P66

Fig. 2. Effect of the growth of the four selected fungal strains in 25% v/v OMW onthe reduction of the medium’s total phenolics during a 30-d incubation period.Error bars represent standard errors of the means (n = 4). Lack of letters in commonindicates statistically significant differences (Tukey’s test, p < 0.05) between treat-ment means at different time-periods for each strain (capital letters), and at thesame time period among the four fungal strains (lowercase letters).

Table 3Determination of changes in pH, electrical conductivity (EC), COD and biotoxicity of25% v/v OMW after 30-d treatment with the four selected fungal strains. Biotoxicity isevaluated as inhibition of luminiscence and it is expressed as ED50 or ED20 valuesobtained with LumisTox test (ED50 and ED20 correspond to 50% and 20% inhibitionrespectively). The data were obtained after a 30 min exposure to the effluent. Highervalues indicate lower toxicity.

OMW treatment pH EC(mS cm�1)

COD(g L�1)

ED20

(%)ED50

(%)

Control 6.16 3.1 12.37 ± 0.92 1.15 5.10Ganoderma australe IK-

16006.86 2.9 8.85 ± 0.18 7.57 26.79

Ganoderma carnosumIK-1642

6.00 2.9 10.06 ± 0.88 8.76 34.35

Pleurotus ostreatusLGMACC 22

7.42 2.5 10.88 ± 0.49 10.58 40.19

Pleurotus eryngii LGAMP66

8.31 2.3 10.48 ± 0.44 16.86 74.93

624 S. Ntougias et al. / Chemosphere 88 (2012) 620–626

4. Discussion

Ganoderma and Pleurotus spp. demonstrated distinct decoloriza-tion patterns, whereas no differences in decolorization were ob-served within the same genus. Hence, Pleurotus spp. invokedsignificant OMW color reduction (by 60–65%); similar values werepreviously reported only among the most efficient white-rot fungiexamined (Jaouani et al., 2003; Dias et al., 2004; Matos et al., 2007).Ganoderma spp. showed a relatively lower OMW decolorization(up to 46%). The initial-temporary increase in color intensity,which was observed for the OMW samples treated with Ganodermaspp., could be attributed to the oxidation and/or polycondensationof phenolics to darker-colored compounds (Thurston, 1994;Jaouani et al., 2006). In addition, laccase oxidative action on phen-olics is held responsible for the formation of unstable radical inter-mediates, which could further lead to rapid polymerization (Farnetet al., 2000; Saparrat et al., 2010). On the other hand, this studydemonstrated that OMW decolorization by Ganoderma strainswas correlated with the reduction of phenolics; it is noteworthythat earlier findings associated OMW color with the presence ofhigh molecular-mass polyphenols (Sayadi et al., 2000). Moreover,

the results of this work revealed a significant correlation betweenlaccase activity over time and OMW decolorization by P. eryngii;hence, the outcome of previous pertinent studies on other white-rot fungi are confirmed (D’Annibale et al., 2004; Dias et al., 2004).

OMW phenolics removal was higher when the effluent wastreated by Pleurotus spp., although their degradation by Ganodermastrains started earlier. Laccase activity in Pleurotus treatments in-creased rapidly following the initial lag-phase and was in accor-dance to phenolic content decrease. A correlation between totalphenolics removal and laccase activity was noted for effluents fromthe green olive debittering-process treated by white-rot fungiincluding P. ostreatus (Aggelis et al., 2002). In addition, a small de-crease in phenolics reduction was observed from the 20–30 d ofthe incubation, most likely indicating the removal of easily degrad-able phenolics and the increasing recalcitrance of the remainingphenolic compounds (Casa et al., 2003) or even the accumulationof excess laccase in aged cultures. In adverse environments, laccaseactivity is induced as an adaptive mechanism of survival for white-rot fungi (Thurston, 1994; Baldrian, 2006); this induction for Pleu-rotus spp. has been reported to be related with the amount ofOMW in the cultivation substrate (Tomati et al., 1991; Tsioulpaset al., 2002). In our study, Pleurotus strains showed a delay at theonset of laccase activity, indicating a slow adaptation in the spe-cific composition of the effluent used. This lag phase in enzymeproduction could be thus attributed to the time needed for theseparticular fungi to adapt their degradation mechanisms to theOMW constituents and to cope with their toxicity (D’Annibaleet al., 2004).

Ganoderma spp. showed high Mn-independent peroxidaseactivity values during the first half of the incubation period,whereas Pleurotus spp. presented higher enzyme activities duringthe last 10–15 d of growth. In contrast, MnP activity (most promi-nent in P. ostreatus) was only detected in the latter stage of OMWdegradation process; this is probably due to the limitation of theeasily degradable phenolics at the end of detoxification period.Moreover, MnP has been reported to be produced as a result ofthe secondary metabolism induced by nitrogen or carbon starva-tion (Kirk and Farrell, 1987), which was apparently not the caseduring the first 10 d of incubation of the four selected strains usedin this study. It is noteworthy that MnIP activity correlated withOMW phenolics removal by P. ostreatus. In addition, MnP and MnIPactivities were detected for both G. australe and G. carnosum; in thepast, the sole Ganoderma species examined for OMW treatmentwas G. applanatum, which however exhibited only laccase activity(Matos et al., 2007). Despite the fact that the enzyme activitiesmeasured provided useful data at elucidating mechanisms ofOMW biodegradation, it is very likely that other factors (not exam-ined here) contribute at the progress of this complex process aswell.

Phytotoxicity tests in the present study demonstrated thatOMW treatment with Pleurotus strains led to increased seed germi-nation values during the second half of the incubation period. Re-cently, Aviani et al. (2009) reported that OMW treatment with P.ostreatus resulted in rather limited phytotoxicity alleviation de-spite the substantial decrease in total phenolics, and they recom-mended the combined use of plant and microbial toxicity assaysat evaluating OMW degradation. On the other hand, Ganodermastrains did not decrease OMW phytotoxicity; noteworthy, treat-ment with G. australe presented higher phytotoxicity (comparedto untreated OMW) at the end of incubation, which could be attrib-uted to the formation of phenoloxidase reaction products (e.g.phenoxy-radicals and quinonoids) that are more toxic than theirprecursors (Saavedra et al., 2006).

Bioluminescence assays employing A. fischeri are widely usedfor routine assessment of effluents toxicity (Ribo, 1997; Jenningset al., 2001). Mekki et al. (2008) confirmed the high sensitivity of

bc, Aa, A

A for controlB for IK-1600A for IK-1642

A for LGAM P66AB for LGMACC 22

a, A

a, B

a, A- controla, B - IK1600

a, A - LGAM P66a, A - LGMACC 22

b, A

a, A

a, A

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ab, AB

d, Bb, B

0

10

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50

60

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80

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3020100

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Ger

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atio

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controlG. australe IK-1600G. carnosum IK-1642P. ostreatus LGMACC 22 P. eryngii LGAM P66

Fig. 3. Effect of the growth of the four selected fungal strains in 25% vv OMW on the reduction of phytotoxicity during a 30-d incubation period. Water was used as control.Error bars represent standard errors of the means (n = 4). Lack of letters in common indicates statistically significant differences (Tukey’s test, p < 0.05) between treatmentmeans at different time-periods for each strain (capital letters), and at the same time period among the four fungal strains (lowercase letters).

S. Ntougias et al. / Chemosphere 88 (2012) 620–626 625

this test in evaluating OMW toxicity following effluent’s electro-coagulation combined with anaerobic digestion or after its applica-tion into soils; results showed significant alleviation of bacteriumgrowth inhibition, especially in soil-treated OMW. The presentstudy examined for the first time the effect of Pleurotus and Gano-derma treated OMW on A. fischeri. All selected strains reducedOMW toxicity by ca. 5–15 times compared to the control. In thepast, pretreatment of OMW by activated sludge bioaugmentedwith P. chrysosporium and Trametes versicolor resulted in a toxicitydecrease by 26–30 percentage units (Dhouib et al., 2006). In addi-tion, the high performance of Pleurotus species (and especially of P.eryngii) in alleviating A. fischeri growth inhibition could be associ-ated with their respective increased ability in decreasing OMWphenolics. Previous studies demonstrated that the OMW toxicitywas essentially due to its high content in low molecular weightaromatic fraction and to synergistic inhibitory effects of phenolics(Mekki et al., 2008; Aviani et al., 2009).

In conclusion, Pleurotus and Ganoderma spp. were adaptedeffectively to grow on 25% OMW and demonstrated high activitiesof OMW degrading enzymes; this resulted in a large decrease ofthe effluent’s toxicity by ca. 5–15 times. The high reduction of totalphenolics content and the significant decolorization (mainly byPleurotus spp.) of OMW indicate the suitability of such organismsto form part of a viable solution for their integrated treatment.Such fungal-based methodologies should be optimized for address-ing specific targets (i.e., phenolics decrease) which are technicallyand economically viable, and they should be combined with otherapproaches (e.g. pre-treatment by chemical methods for loweringthe amount of suspended solids, and post-treatment throughlarge-scale anaerobic processes for reducing COD values) in orderto arrive at an environmentally-sound OMW management.

Acknowledgements

This study was partly funded by the Joint Research and Technol-ogy Programme between Greece and the Czech Republic entitled‘‘Integrated treatment and remediation of recalcitrant agriculturaland industrial wastes with high content in polyphenolics and dyes’’(Greek General Secretariat of Research and Technology, andMinistry of Education, Youth and Sports of the Czech Republic).PB, FN and VM were supported by the Institutional Research Con-cept of the Institute of Microbiology of the ASCR (AV0Z50200510).

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