Appl Microbiol Biotechnol (2002) 59:353–360 DOI 10.1007/s00253-002-1005-9
O R I G I N A L PA P E R
G. Aggelis · C. Ehaliotis · F. Nerud · I. Stoychev G. Lyberatos · G. I. Zervakis
Evaluation of white-rot fungi for detoxification and decolorization of effluents from the green olive debittering process Received: 7 December 2001 / Revised: 11 March 2002 / Accepted: 13 March 2002 / Published online: 4 May 2002 © Springer-Verlag 2002
Abstract Wastewater produced by the debittering process of green olives (GOW) is rich in polyphenolics and presents high chemical oxygen demand and alkalinity values. Eight white-rot fungi (Abortiporus biennis, Dichomitus squalens, Inonotus hispidus, Irpex lacteus, Lentinus tigrinus, Panellus stipticus, Pleurotus ostreatus and Trametes hirsuta) were grown in GOW for 1 month and the reduction in total phenolics, the decolorization activity and the related enzyme activities were compared. Phenolics were efficiently reduced by P. ostreatus (52%) and A. biennis (55%), followed by P. stipticus (42%) and D. squalens (36%), but only P. ostreatus had high decolorization efficiency (49%). Laccase activity was the highest in all of the fungi, followed by manganese-independent peroxidase (MnIP). Substantial manganese peroxidase (MnP) activity was observed only in GOW treated with P. ostreatus and A. biennis, whereas lignin peroxidase (LiP) and veratryl alcohol oxidase (VAOx) activities were not detected. Early measurements of laccase activity were highly correlated (r2=0.91) with the final reduction of total phenolics and could serve as an early indicator of the potential of white-rot fungi to efficiently reduce the G. Aggelis · C. Ehaliotis (✉) · G.I. Zervakis National Agricultural Research Foundation, Institute of Kalamata, Lakonikis 85, 24100 Kalamata, Greece e-mail:
[email protected] Tel.: +30-7210-91984, Fax: +30-7210-27133 G. Aggelis · G. Lyberatos University of Patras, Department of Chemical Engineering, Laboratory of Biochemical Engineering and Environmental Technology, Panepistimioupoli, Rion 26500, Greece C. Ehaliotis Agricultural University of Athens, Department of Natural Resources and Agricultural Engineering, Laboratory of Soils and Agricultural Chemistry, Iera Odos 75, 11855 Athens, Greece F. Nerud · I. Stoychev Academy of Sciences of the Czech Republic, Institute of Microbiology, Laboratory of Biochemistry of Wood-Rotting Fungi, Videnska 1083, 14220 Prague 4, Czech Republic
amount of total phenolics in GOW. The presence of MnP was, however, required to achieve efficient decolorization. Phytotoxicity of GOW treated with a selected P. ostreatus strain did not decline despite large reductions of the phenolic content (76%). Similarly, in GOW treated with purified laccase from Polyporus pensitius, a reduction in total phenolics which exceeded 50% was achieved; however, it was not accompanied by a decline in phytotoxicity. These results are probably related to the formation of phenoxy radicals and quinonoids, which re-polymerize in the absence of VAOx but do not lead to polymer precipitation in the treated GOW.
Introduction Liquid effluents generated by olive-processing plants constitute an issue of significant environmental concern for all major olive-producing areas, including most Mediterranean countries. Although the table-olive industries yield smaller quantities of wastewater than olive-oil mills, their safe disposal presents severe problems, mainly due to their recalcitrance and the seasonality of production. World table-olive production has recently reached an all time high of 1,225,500 tons (International Olive Oil Council 2000); their post-harvest treatment results in an effluent volume of 3.9–7.5 m3 ton–1 for green olives and 0.9–1.9 m3 ton–1 for black olives (Kopsidas 1992). In particular, wastewater resulting from the debittering of green olives (GOW), through their treatment with sodium hydroxide (1.8–2.3% w/v), is rich in polyphenols and organic acids, and has high COD and alkalinity values. Most of the wastewater is released into water receptors without prior treatment, or sent directly to stabilization ponds, causing serious pollution and odor emission problems. The biotoxic properties of GOW can be partly attributed to its phenolic content, as demonstrated for olive-mill effluents (Ehaliotis et al. 1999; Gonzalez et al. 1990; Moreno et al. 1987; Perez et al. 1986, 1992). This may not only hinder direct application in natural receptors but could also inhibit successful
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application of conventional aerobic (Benitez et al. 1997; Knupp et al. 1996) or anaerobic (Beccari et al. 1999; Borja et al. 1996; Sierra-Alvarez and Lettinga 1990) bioremediation processes. In the past, various physicochemical methods aimed at reducing the organic load and the toxicity of GOW have been applied at laboratory-scale. Precipitation, adsorption on activated carbon, and ultrafiltration have all been investigated (Brenes and Carrido 1988; Brenes et al. 1990; Gomez-Millan et al. 1983), while ozonation (Beltran et al. 1998) and oxidation by Fenton’s reactions (Filipakopoulou et al. 1999) have also been employed, demonstrating significant COD removal and reduction of polyphenol content. However, scaling-up of such processes has often met serious technical difficulties and long-term economic failure. White-rot fungi have been successfully used for the biodegradation and bioremediation of a large array of organic pollutants, including polycyclic aromatic hydrocarbons, DDT, TNT, polychlorinated aromatic compounds, and synthetic dyes (Bumpus et al. 1985; Cerniglia 1992; Fernando et al. 1990; Lang et al. 1996; Michel et al. 1991; Mileski et al. 1988; Pointing 2001; Rodriguez et al. 1999). Their enzymatic systems are capable of oxidative depolymerization and subsequent mineralization of lignin-related compounds (Ander and Eriksson 1978; Kirk 1984; Nerud et al. 1991). Therefore, several species have been recently evaluated as regards their efficiency to bioconvert agroindustrial wastes that are rich in polyphenols. Examples of such approaches for the treatment of olive-oil mill wastewater include the use of the extensively studied fungus Phanerochaete chrysosporium for decolorization (SaizJimenez and Gomez-Alarcon 1986; Sayadi and Ellouz 1992, 1995), Pleurotus spp. for COD reduction, decolorization, edible biomass or mushroom production (Sanjust et al. 1991; Setti et al. 1998; Zervakis and Balis 1996a; Zervakis et al., 1996), and Lentinula edodes for the formation of metabolites (Grapelli et al. 1991). However, despite the research interest that treatment of olive-oil mill wastewater has attracted, relevant studies on GOW are practically non-existent (Aggelis et al. 1999). The objective of this study was to compare several species of white-rot fungi for their ability to decrease the phenolic content of GOW and to decolorize GOW, and to relate bioremediation to the principal enzyme activities detected. The efficiency of a selected Pleurotus ostreatus strain to modify the polluting properties of GOW was then further exploited.
Materials and methods Organisms Eight white-rot fungi (class Basidiomycotina), i.e. Abortiporus biennis (CCBAS 521), Dichomitus squalens (CCBAS 751), Inonotus hispidus (CCBAS 810), Panellus stipticus (CCBAS 450), Pleurotus ostreatus (CCBAS 472), Trametes hirsuta (CCBAS 610), Irpex lacteus (CCBAS 238), and Lentinus tigrinus (CCBAS 617/93) were used for the first set of experiments, which involved measurements of the reduction of total phenolics, the decolorization and the enzyme activities in batch cultures of GOW.
Table 1 Physicochemical characteristics of wastewaters resulting from the debittering of green olives (GOW). TSS total suspended solids, VSS volatile suspended solids, COD chemical oxygen demand, TOC total organic carbon, TKN total Kjeldahl nitrogen, VFAs volatile fatty acids pH Electrical conductivity (mS/cm2) TSS (mg/l) VSS (mg/l) TOC (mg/l) Total COD (mg/l) Dissolved COD (mg/l) Total phenolics (mg/l) TKN (mg/l) Total phosphorus (mg/l) Total VFAs (mg/l) Na (mg/l) K (mg/l) Ca (mg/l) Mg (mg/l) Cl (mg/l) Fe (mg/l) Zn (mg/l) Mn (mg/l) Cu (mg/l) S (mg/l) Ni (mg/l) Co (mg/l)
12.06 13.00 3420 2860 24500 26720 24263 1384 500 38.39 528 3100 2300 248.248 52.70 350 1.86 3.23 0.42 3.80 345 0.24 0.14
Sixteen Pleurotus strains (selected out of a preliminary Petri dish screening involving 45 strains) were evaluated in a second set of experiments in GOW: P. ostreatus (IK P69, IK P61, IK P72, IK P59, IK P57, ATCC 34675), P. pulmonarius (IK P46, IK P41, IK P12, IK P10, CBS 13285), and P. eryngii (IK P109, IK P101, IK P63, UPA 12, ATCC 36047). The strain abbreviations used are: NAGREF-IK: National Agricultural Research Foundation, Institute of Kalamata, Greece; CCBAS: Academy of Sciences of the Czech Republic, Institute of Microbiology, Prague; ATCC: American Type Culture Collection, Rockville, USA; CBS: Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands; UPA: University of Palermo, Department of Botany, Italy. All strains are maintained at the fungal Culture Collections of NAGREF-IK, and CCBAS. Media and culture conditions The GOW was obtained from the Arta Agricultural Cooperative olive processing plant (Epirus, Greece) and was stored at 4 °C. An analysis of its physicochemical properties and composition is presented in Table 1. Prior to biological treatment, GOW was diluted by the addition of equal volumes of distilled water (50%, v/v) and the pH was adjusted to 6.0 with H3PO4 (85%, v/v). GOW was subsequently centrifuged for 5 min at 4,250 g (20 °C) and used as substrate in all experiments. Prior to use GOW was heatsterilized (121 °C, 1.2 atm, 20 min). Solidified medium was prepared by the addition of agar (1.6% w/v) to GOW. Inocula originated from the actively growing part (periphery) of fungal colonies cultured in Petri dishes on complete yeast medium at 28 °C (Raper et al. 1972). The fungi were first cultivated on solidified GOW medium. Agar plugs (diam. 6 mm) were then used to inoculate static liquid cultures of 100 ml GOW, which were subsequently incubated at 28 °C. Non-inoculated liquid GOW media were also incubated and used as controls. Mycelium linear growth rates and decolorization on GOW solid-state cultures In preliminary screening experiments of Pleurotus strains, mycelium growth rates were measured in Petri dishes (9 cm) containing
355 15 ml of solidified GOW medium. Inocula (diam. 6 mm) were transferred from the periphery of 5-day-old colonies growing on GOW and were placed into the center of the dishes. Linear growth rates were determined as the average of the distance covered by the mycelium front along two perpendicular directions (Zervakis and Balis 1996b); measurements were taken every 24 h for 8 days, at 28 °C. The degree of decolorization of solidified GOW media was established by visual inspection. Mycelium dry weights Liquid fungal cultures were incubated for 1 month; at regular intervals three flasks were removed to perform the measurements described below. Mycelium weights were determined following harvesting of liquid cultures, filtration under vacuum, and drying onto filter paper discs at 80 °C for 24 h. Enzymes and assays Enzyme activities were estimated spectrophotometrically. Laccase (E.C. 1.10.3.2: benzenediol: oxygen oxidoreductase) activity was determined at 425 nm by monitoring the oxidation of 0.4 ml ABTS (2,2-aminobis(3-ethylbenzothiazoline-6-sulphonic acid)) (1.5 mM) added to 0.8 ml of sample in 1.2 ml Na-tartrate buffer (0.1 M, pH 4.5) (Bourbonnais and Paice 1990). Manganeseindependent peroxidase (MnIP) was determined at 590 nm by the oxidative coupling of 0.1 ml MBTH (3-methyl-2-benzothiazoline hydrazone) (1 mM) and 0.2 ml DMAB (3-dimethylaminobenzoic acid) (25 mM) added to 0.66 ml sample in 1 ml succinate-lactate buffer (0.1 M, pH 4.5) in the presence of 0.01 ml H2O2 (10 mM), after subtracting background activity (determined as above but in the absence of H2O2). Manganese peroxidase (MnP, E.C. 1.11.1.13 (MnII): hydrogen-peroxide oxidoreductase) activity was determined by the same method used for MnIP following the addition of Mn2+ ions (0.01 ml MnSO4, 20 mM) after subtracting MnIP activity (Ngo and Lenhoff 1980). Lignin peroxidase (LiP, E.C. 1.11.1.14: diarylpropane: oxygen, hydrogen-peroxide oxidoreductase) activity was determined at 310 nm by monitoring the oxidation of 0.07 ml veratryl alcohol (20 mM) to veratryl aldehyde added to 0.8 ml sample in 1.6 ml Na-tartrate buffer (0.1 M, pH 3) in the presence of 0.03 ml H2O2 (54 mM) (Tien and Kirk 1984). Veratryl alcohol oxidase activity (VAOx) was determined as for LiP, but in the absence of H2O2. In all cases, one activity unit was defined as the amount of enzyme transforming 1 µmol of substrate min–1. Purified laccase from Polyporus pensitius (Novoferm 122, Novozymes, Switzerland) was added directly to 25-ml aliquots of GOW, pre-treated as described above. The aliquots were initially agitated vigorously for 15 min (180 rpm) and mildly (90 rpm) thereafter. Samples were taken for analysis at 1-h, 2-h, 20-h and 40-h intervals. Content of total phenolics The concentration of total phenolics was determined by the FolinCiocalteu method (Waterman and Mole 1994), based on the ability of phenolics to reduce phosphomolybdic-phosphotungstic reagent as evidenced by the formation of a blue complex that can be determined spectrophotometrically at 760 nm. The phenolic content of the samples was expressed as syringic acid equivalents (10 µg ml–1 gives an optical density of 0.377 at 760 nm). Decolorization After harvesting mycelium from the liquid cultures, decolorization of the filtered GOW broth was assessed spectrophotometrically by measuring absorbance at 525 nm (Martirani et al. 1996).
Chemical oxygen demand Dissolved chemical oxygen demand (COD) was determined as proposed by standard methods for the examination of water and wastewater (American Public Health Association 1995). Phytotoxicity The phytotoxicity of GOW liquid media was assessed by measuring the germination and root elongation of Lepidium sativum seeds. Triplicates of 25 seeds were placed for germination for 3 days at 28 °C on filter paper (Whatman #1), pre-soaked in the GOW substrate to be examined. Germination indices were calculated according to Zucconi et al. (1981). Statistical analysis For all experiments, three replicates per strain were used unless otherwise stated. Standard errors of mean values (S.E.) were calculated, and simple, stepwise and backward multiple linear regression analysis was performed using SPSS software (version 6 for Windows OS).
Results and discussion Biodegradation and related enzyme activities of eight white-rot fungi growing on GOW All of the white-rot fungi initially studied (Abortiporus biennis, Dichomitus squalens, Inonotus hispidus, Irpex lacteus, Lentinus tigrinus, Panellus stipticus, Pleurotus ostreatus and Trametes hirsuta), grew well in batch cultures of GOW. P. ostreatus showed the highest biomass production (94.1±4.2 mg l–1 increase in dry weight within 30 days), followed by L. tigrinus (55.5±3.1 mg l–1). The lowest dry weight production was observed for I. lacteus (10.1±3.2 mg l–1) and A. biennis (10.9±3.1 mg l–1), while the other four species showed intermediate growth (29.7–43.3 mg l–1). Most of the fungi growing in GOW liquid cultures reached maximal growth at day 16, with the exception of Panellus stipticus which reached maximal growth at day 22, and Pleurotus spp. which grew until the end of the cultivation period. Under the tested conditions, the most efficient species, as regards reduction of total phenolics, were P. ostreatus and A. biennis, reaching 51.5% and 54.5% reduction of the initial phenolics content, respectively (Fig. 1a), followed by P. stipticus and D. squalens (42.2% and 36.4%, respectively). The other four fungi (T. hirsuta, I. hispidus, L. tigrinus and I. lacteus) did not demonstrate significant degradation of phenolics during the incubation period. P. ostreatus and A. biennis exhibited the same pattern of reduction of total phenolics, with degradation beginning during the first days of incubation and reaching a maximum rate between days 7 and 22 (Fig. 1a). Due to its limited mycelial growth, A. biennis exhibited the highest reduction of phenolics per unit dry weight. The relationship between overall reduction of phenolics and decolorization was poor (r2=0.561, P=0.03). Only P. ostreatus showed a high degree of GOW decolorization
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Fig. 2 Coefficients of determination (r2) obtained by regression analysis between the enzyme activities of the eight white-rot fungi at different incubation times vs the reduction of phenolics achieved by these fungi at the end of a 31-day incubation in GOW
Fig. 1 Effect of eight white-rot fungi growing in wastewater resulting from the debittering of green olives (GOW) on a the reduction of total phenolics, and b decolorization, during a 31-day incubation period. Error bars Standard errors of the means (n=3)
(48.9%) followed by the substantially less efficient A. biennis and P. stipticus (9.1% and 8.4%, respectively, Figure 1b). The rest of the species examined did not decolorize the wastewater. The slow color removal by I. lacteus observed during the initial stages of incubation subsequently ceased, apparently due to the production of metabolites absorbing light at 525 nm. This was also observed for L. tigrinus, T. hirsuta, I. hispidus and D. squalens, which showed increasing absorbance values with time. The production of lignolytic enzymes started near the end of the exponential phase of growth or at the beginning of the stationary phase of growth of the fungi. Laccase
activity was the highest in all of the cultures, followed by MnIP (Table 2). P. ostreatus and A. biennis demonstrated much higher laccase activity than the rest of the species examined, followed by P. stipticus and D. squalens. These four species (the same that drastically reduced phenolics in GOW) showed significant laccase activity already during the first ten days of incubation. Indeed, the overall decrease in phenolics (at the end of incubation) was strongly correlated with laccase activity for the respective eight fungi at early stages of incubation (Fig. 2). Laccase production could, therefore, be used as an early indicator of the potential of white-rot fungi to lower the phenolics content in GOW. Correlation of the reduction in overall phenolics with laccase activities measured at later incubation stages was slightly poorer (Fig. 2), probably due to the increasing recalcitrance of the remaining phenolic compounds or to the accumulation of excess laccase in the older cultures of the efficient fungi only. Differences in MnIP activity were less dramatic, but P. ostreatus, A. biennis, P. stipticus and D. squalens still had the highest activity levels (Table 2). MnIP activity correlated more weakly with the total phenolics removal, and this was recorded only towards the end of the incubation period (Fig. 2). Substantial MnP activity was only observed in P. ostreatus and, at a later stage, in A. biennis (Table 2) and did not correlate significantly with total phenolics removal at any phase of the incubation period (Fig. 2). LiP and VAOx activities were not detected in any of the species tested. The absence of any LiP activity in any of the fungi examined in this study may explain the relatively low decolorization efficiencies observed, since high levels of
357 Table 2 Laccase, manganese peroxidase, and manganeseindependent peroxidase enzyme activities exhibited by eight white-rot fungi during a 31-day incubation period (mean values±standard errors, n=3, errors not shown are ≤0.3)
Table 3 Removal of phenolics and decolorization of GOW during incubation with purified laccase from Polyporus pensitius (standard error values are 0.96 for the first 3 weeks of incubation, with best correlation obtained for day 13: %final decolorization=1.141 laccased13+7.448 MnPd13, R2=0.982). The inefficiency of laccase alone to decolorize
Panellus stipticus
Pleurotus ostreatus
Trametes hirsuta
0.5 8.8±0.5 15.4±0.5 24.3±2.2 29.7±1.0
6.3 16.4±1.9 36.6±1.4 60.1±2.8 67.3±2.1
0.0 0.9 2.8 5.4±0.4 6.2
0.6 5.3 14.5±1.5 16.8±1.3 19.1±0.9
0.0 0.0 0.2 0.0 0.4
0.0 0.1 0.2 0.5 0.1 0.3 1.8 4.3±0.7 11.2±0.6 12.5±0.8
0.3 2.1 8.6±0.7 14.4±1.3 16±0.6
0.0 1.8 3.4±0.5 10.1±1.2 10
0.264 µl lac ml–1
1.32 µl lac ml–1
2.64 µl lac ml–1
4 µl lac ml–1
0.88 0.64 0.61 0.42 0.41
0.88 0.57 0.56 0.43 0.39
0.88 0.55 0.53 0.40 0.36
0.88 0.53 0.48 0.41 0.37
0.49 0.67 0.64 0.52 0.48
0.49 0.65 0.61 0.50 0.46
0.49 0.63 0.60 0.49 0.45
0.49 0.62 0.59 0.49 0.46
GOW is also supported by the results of short-term incubations of GOW with purified laccase from Polyporus pensitius, which resulted in an initial increase in absorbance followed by poor, if any, decolorization despite a reduction in phenolics exceeding 50% (Table 3). Selection and evaluation of an efficient P. ostreatus strain Since P. ostreatus had the highest reduction of phenolics and decolorization efficiency, a broad intra-genus screening was performed among 45 Pleurotus strains originally assigned in three species (P. ostreatus, 18 strains; P. pulmonarius, 12 strains; P. eryngii, 15 strains). Comparative assessment was based on the linear growth rates of the strains and their decolorization efficiency on solidified GOW medium (data not presented). The 16 most efficient strains from Pleurotus ostreatus (6 strains), Pleurotus pulmonarius (5 strains) and Pleurotus eryngii species (5 strains) were further incubated for 30 days in batch cultures with GOW. P. ostreatus strains consistently pro-
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Fig. 3 Degradation of total phenolics and decolorization of GOW by Pleurotus ostreatus (strain IK P69) during a 30-day incubation period. Error bars Standard errors of the means (n=3)
duced the fastest growth (0.181±0.004 cm h–1) and the highest and least variable degradation of phenolics (71.7±1.35%), as compared to strains of P. pulmonarius (0.134±0.005 cm h–1 and 64.7±6.55%, respectively) and P. eryngii (0.075±0.002 cm h–1 and 45.1±9.68,% respectively). The best strain, P. ostreatus IK P69, was selected and further examined for efficient treatment of GOW. At the end of the 30-day incubation period mycelium production had reached 150 mg l–1, total phenolics were reduced by 76% whereas color had decreased by 37.2% (Fig. 3). LiP and VAOx activities were again not detected and COD reduction was small (12.3%). This is in accordance with the limited degradation of lignocellulosic wastes reported for P. ostreatus (Kerem et al. 1992). By contrast, Phanerochaete chrysosporium efficiently degraded organic matter in lignocellulosic wastes (Kirk and Farell 1987) and reduced the COD in olive-oil mill wastewater (Sayadi and Ellouz 1992; Kissi et al. 2001). LiPs produced by P. chrysosporium are capable of catalyzing the oxidation of a broad range of aromatic hydrocarbons (Kirk and Farell 1987), whereas laccases oxidize mainly phenolic compounds and may oxidize non-phenolic compounds only in the presence of mediator substrates (Bourbonnais and Paice 1990; Breen and Singleton 1999). The inefficient COD reduction in GOW by P. ostreatus could therefore be related to the lack of a typical LiP in Pleurotus (Caramelo et al. 1999; Valmaseda et al. 1991), although there are reports of versatile Pleurotus peroxidases, sharing catalytic properties of LiP and MnP, which enable them to act on Mn2+, simple phenols and veratryl alcohol (Camarero et al. 1999; Ruiz-Duenas et al. 1999).
Incubation with P. ostreatus did not result in any decline of phytotoxicity in the GOW, despite the large reduction in phenolics and the observed decolorization. The lack of germination of Lepidium sativum in 50% GOW at T0 was still observed at the end of incubation with P. ostreatus (T30); when the medium was diluted 1:1 with water the germination index increased non-significantly from 15% to 16%. When 50% filter-sterilized GOW was used (0.2-µm microcellulose filter, Nalgene, USA) the increase of the germination index at the end of incubation, from 32% to 36%, was again non-significant. Sodium (Na+) is a major element in the composition of GOW (originating from the standard debittering treatment of the green olives with NaOH). When water was brought up to the original electrical conductivity of GOW using NaCl, the germination index was reduced to 66.7% as compared to control water (100%). However the original GOW showed a much greater reduction of the germination index (to 14.5%). Similarly, the germination index for diluted GOW (1:1 in water) was only marginally reduced (from 37.6% to 29.5%) when the medium was brought up to 100% of the electrical conductivity of GOW. The results indicate that salinity is only partly responsible for the phytotoxic effects of the wastewater. Since the fungus greatly reduced the phenolics content (76%) but did not decrease the phytotoxicity of GOW, the modified phenolic fraction does not appear to contribute significantly to the phytotoxic properties of GOW. The reduction in phenolics observed following the addition of purified laccase from Polyporus pensitius to GOW (Table 2) was also not accompanied by a decline in phytotoxicity. This is in contrast to the olive-oil mill wastewater treatment, in which incubation with Pleurotus spp. significantly decreased its phytotoxic properties (Martirani et al. 1996; Zervakis et al. 1996). These results indicate that phytotoxicity of agro-industrial wastewaters rich in phenolics may not always be directly related to their total phenolics content. In the GOW treatments described in this work, the predominance of laccases would be expected to produce phenoxy radicals and quinonoids which are known to repolymerize in the absence of VAOx (Marzulo et al. 1995). It has been reported that a major detoxification mechanism following treatment with laccases is the formation of polyphenolic precipitates, which allows for complete physical removal of certain phenolics from the medium (Bollag et al. 1988). In the absence of precipitate formation, the oxidation of phenolics by laccase may not alter their toxicity (Bollag et al. 1988) or may even increase it (Haars and Hutterman 1980). Therefore, the inefficient detoxification of GOW could be attributed to the absence of polymer precipitation in treated GOW. In conclusion, several white-rot fungi from different genera greatly reduced the content of phenolics in GOW, but only Pleurotus species demonstrated high decolorization efficiency. Early laccase activity was strongly correlated with the overall reduction of phenolics, and laccase alone efficiently reduced phenolics when
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added to GOW in a purified form. However, laccase activity did not reduce phytotoxicity, an effect probably related to the lack of polymer precipitation, whereas the production of MnP was apparently needed to induce decolorization, perhaps by advancing the oxidation of nonphenolic compounds. LiP and VAOx activities were not produced by any of the fungi examined and the poor COD reduction is in line with the lack of LiP production. Acknowledgements We are grateful to Maria Tourna for excellent technical assistance and to Kalliopi Papadopoulou for helpful suggestions and valuable comments on the manuscript. Thanks are extended to Dr. Anne Osbourn for critical reading of the manuscript. The gift of Novoferm 122 from Novozymes AG is greatly appreciated. This work was partially funded by the National Agricultural Research Foundation (Greece), and supported by the Institutional Research Concept AVOZ 5020903, Institute of Microbiology, ASCR, Prague.
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