Journal of Biotechnology 61 (1998) 209–218

The biodegradation of recalcitrant effluents from an olive millby a white-rot fungus

Alessandro D’Annibale *, Claudia Crestini *, Vittorio Vinciguerra,Giovanni Giovannozzi Sermanni

Department of Agrobiology and Agrochemistry, Tuscia Uni6ersity, Via San Camillo de Lellis s.n.c., 01100 Viterbo, Italy

Received 22 February 1997; received in revised form 16 February 1998; accepted 18 February 1998

Abstract

Biodegradation of olive-mill wastewater (OMW) was performed by the polyurethane-immobilized mycelium ofLentinula edodes. Throughout three consecutive treatment cycles of the effluent significant abatements of its pollutingcharacteristics were observed. In fact, its contents in total organic carbon, total phenols, total ortho-diphenol weredramatically reduced. In addition, a significant effluent decolorization was evident. © 1998 Elsevier Science B.V. Allrights reserved.

Keywords: Wastewater; Phenolic effluents; White-rot fungi; Decolorization; Lentinula edodes ; Ligninolytic enzymes

1. Introduction

In the Mediterranean area, olive oil productiongenerates more than 3×107 m3 of an effluentdesignated olive-mill wastewater (OMW) (Sayadiand Ellouz, 1995). This effluent is characterizedby a high polluting load, due to its high contentof organic substances, and because of a hightoxicity towards several biological systems(Peredes et al., 1986; Moreno et al., 1987; Capassoet al., 1992). The toxicity of the effluent has been

attributed to its phenolic constituents, which con-stitute from 1.5 to 8 g l−1, and is strictly depen-dant on the processing system used for olive oilproduction. A significant aliquot of these phenolicmaterials is represented by the monomeric frac-tion (Vasquez et al., 1974, Capasso et al., 1992).Another relevant problem associated with thiseffluent is its strong dark colour, which is mainlydue to chromophoric lignin-related materials withdifferent degrees of polymerization (Sayadi andEllouz, 1992).

Several physico-chemical processes, includingsimple evaporation (Storm, 1989), reverse osmosisand ultrafiltration (Reimers Suarez, 1983) have

* Corresponding authors. Tel.: +39 761 357234; fax: +39761 357242; e-mail: Crestini@unitus.it; Dannib@unitus.it

0168-1656/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

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been employed in order to detoxify this effluent.These solutions require costly investment andmaintenance which conflict with the seasonalcharacter of the olive-oil extraction industry.Therefore, great interest has been focused on bio-logical treatment of OMW, as an alternative tothe conventional treatment processes. The biologi-cal disposal of this wastewater by anaerobic diges-tion has been investigated by several authors(Fiestas et al., 1982, Rozzi et al., 1989; Martin etal., 1991), but inhibitory effects towardsmethanogenic bacteria caused by high concentra-tions of aromatic compounds have been reported(Boari et al., 1984). The problem has also beenpartially solved by diluting the waste, and recentlyit has been shown that a previous aerobic treat-ment of OMW with Aspergillus niger (Borja et al.,1995) or with Geotrichum candidum enhances theprocess of anaerobic digestion, due to the removalof phenolic inhibitors (Borja et al., 1992). In thiscontext, the use of fungi characterized by aproved lignolytic efficiency and, therefore, by ahigh capability of degrading lignin-related com-pounds, such as polyphenols, represents a promis-ing pespective (Archibald et al., 1990; Bergabueret al., 1991; Sayadi and Ellouz, 1992). The lignin-degrading ability of white-rot fungi seems to beassociated with the release of extracellular en-zymes, which mainly include lignin-peroxidase,Mn-dependent peroxidase and laccase (Tuor etal., 1995). The white-rot fungus L. edodes pro-duces lignolytic enzymes and degrades lignin dur-ing the primary vegetative growth (Leatham,1986). Its enzymatic complex mainly includes Mn-dependent peroxidase and laccase (Buswell et al.,1995). The latter enzyme is produced after a fewdays of growth and its production is not inhibitedby a high carbon and/or nitrogen content of themedium (Buswell et al., 1995, Crestini et al.,1996a). Previous studies performed on OMW withL. edodes demonstrated its effectiveness in thedegradation of the effluent and revealed that thefungus was able to carry out a significant decol-orization in the presence of a readily availablecarbon source like glucose and without air oroxygen purging (Vinciguerra et al., 1995). More-over, it was shown that ortho-diphenols weredegraded earlier than other phenolic constituents

(Vinciguerra et al., 1995), such as the monophenoltyrosol, whose biotransformation products havebeen recently investigated (Vinciguerra et al.,1997). The affinity of a purified laccase isozymefor this monomer and for the meta-diphenol re-sorcinol, another recalcitrant phenolic monomerof OMW, was found to be lower at least by oneorder of magnitude than the other monomers(D’Annibale et al., 1996a).

Several studies have reported the use of immo-bilization protocols applied to white-rot ba-sidiomycetes (Livernoche et al., 1983; Eaton et al.,1982). The use of these ligninolytic fungi in immo-bilized form turned out to be a promising ap-proach, since it opened the way to the possibilityof mycelial biomass recycling in effluent treatmentapplications (Livernoche et al., 1981).

The aim of the present study was to assess thepossibility of using the polyurethane-sponge-im-mobilized mycelium of L. edodes in the biodegra-dation of olive mill wastewater. This study alsoprovides evidence that the depolymerization ofthe high molecular weight fraction as well as themaximum extent of phenol removal in the effluentrequires the simultaneous presence of laccase andMnP, although the degradation of this fraction isnot necessarily associated with the extent ofeffluent decolorization.

2. Materials and methods

2.1. Materials

Polyurethane sponge (0.4–0.6 mm pore size)was cut into :1.2-cm cubes, which were washedand autoclaved in deionized water before use.Olive mill wastewaters (OMW) were obtainedfrom a mill located in Viterbo, which used acontinous solid–liquid centrifuge processingsystem.

2.2. Microorganism and inoculum preparation

L. edodes (strain SC-495), kindly provided byDr T. Quimio (Los Banos, Philippines), was rou-tinely subcultured on potato-dextrose agar slants.The inoculum was prepared as previously de-scribed (Di Lena et al., 1994).

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2.3. Immobilization of L. edodes

Cultures for immobilization contained 30 1-cmcubes of sponge (1.38 g dry weight) autoclaved in200 ml of the medium used for the inoculum in 2-lErlenmeyer flasks. Inoculation was performedwith 5 ml of inoculum (equivalent to ca. 0.05%(w/v) inoculum) and cultures were incubated at28°C on a gyratory shaker (200 rpm; 2.5 cmradius) for up to 5 days until the surface ofsponge appeared to be uniformly colonized, asdetermined by visual inspection. After this timethe non-adsorbed mycelium was removed by threerepeated washings with sterile deionized water.

Under these conditions the average immobiliza-tion yield was 8% (w/w) as estimated by gravimet-ric analysis (Kirkpatrick et al., 1990).

2.4. Aerobic treatment of oli6e mill waste water

Olive-mill wastewater were centrifuged(11000×g, 30 min), supplemented with saccha-rose (10 g l−1), yeast extract (1 g l−1) and Tween80 (0.05%) and filter-sterilized (0.2 mm). Then,aerobic treatment of OMW was performed byrepeated batch fermentations in 2-l Erlenmeyerflasks using the following standard conditions:effluent 300 ml, temperature 30°C, orbital shaking(30 rpm; 2.5 cm radius), treatment time 8 days foreach batch. Under these conditions the cubes wereonly partially immersed in the liquid medium. Thecultures were purged daily (10 min) with filter-sterilized oxygen provided by a gas-permeablesilicone tubing fitted with a sparger (aeration rate0.35 l min−1). After each batch the carrier waswashed three times with deionized water and sup-plemented-OMW was again added. All the resultswere obtained using triplicate cultures and areexpressed as the mean9S.D.

2.5. Analytical methods

The isolation of the monomeric fraction fromOMW was performed by continuos extractionwith dichloromethane during 24 h, followed bydrying over anhydrous sodium sulphate and sol-vent evaporation.

GC-MS analyses were performed on samplesderivatized with N,O-bis(trimethylsi-lyl)trifluoroacetamide (BSTFA) in pyridine, byuse of a HP GC-MS 5972 spectrometer equippedwith a column HP-5MS 0.25 mm, 30 m, at 70–250°C with an isothermal program at 70°C for thefirst 1 min, then 10°C min−1 and finally isother-mal at 250°C for 20 min. The identification of theproducts was based on the comparison with theretention times and mass spectra of commerciallyavailable pure standards.

Decolorization, total phenols, total organic car-bon and reducing sugars were determined as pre-viously described (Vinciguerra et al., 1995).Ortho-diphenols were quantitated by the molib-date/sodium nitrite (Arnow’s reagent) method(Vasquez et al., 1974). Changes in the apparentmolecular weight fraction of the water-solublepolymeric fraction of OMW was analyzed byhigh-performance size-exclusion chromatography.Duplicate samples (10 ml) were injected into aPWXL-G 3000 column (Supelco, BellefonteUSA), previously equilibrated in 50 mM LiCl(flow rate 1 ml min−1). The column was cali-brated with polymeric standards. Oxalate wasquantitated by using an enzymatic kit fromBoehringer Mannheim. Hydrogen peroxide wasdetermined using a peroxidase-coupled assay, aspreviously described (Giovannozzi Sermanni etal., 1995). Both hydrogen peroxide and oxalatewere tested on the permeate after ultrafiltration at4°C of OMW samples on a YM 5 membrane(Amicon, Danvers) (cut off 5 kDa). This wasmade in order to eliminate the interferences dueto the presence of fungal oxidases.

2.6. Enzymatic assays

Laccase activity was determined spectrophoto-metrically at 477 nm, using 2,6-dimethoxyphenol(e=14600 M−1 cm−1) as a substrate (Slomczyn-ski et al., 1995). Catalase (0.05 U) was added tothe reaction mixture prior to substrate addition,in order to remove traces of hydrogen peroxidepresent in the crude extract. One unit was definedas the amount of enzyme producing 1 mmolmin−1 of product (3,3%,5,5%-tetramethoxydipheno-quinone). Mn-dependent peroxidase activity was

A. D ’Annibale et al. / Journal of Biotechnology 61 (1998) 209–218212

determined by the increase in absorption at 270nm, caused by the formation of Mn(III)-malonate(e=11590 M−1 cm−1) (Waarishi et al., 1992).

3. Results

3.1. OMW decolorization by L. edodes

Undiluted olive-mill wastewater, whose compo-sition is shown in Table 1, were subjected toaerobic treatment with L. edodes previously im-mobilized on polyurethane sponge. OMW wassupplemented with saccharose, a readily-availablecarbon source cheaper than glucose, since it hasbeen shown that the decolorative metabolism re-quires the presence of carbohydrates (Livernocheet al., 1981; Archibald et al., 1990; Bergabuer etal., 1991; Martin and Manzanares, 1994).

Fig. 1 shows the decolorization profiles ob-tained throughout three repeated cycles. The max-imum amount of colour removal was observed atthe end of the first and second cycle of treatment,where 75 and 72% of decolorization was evidentand no initial lag phase was observed as reportedby other authors (Sayadi and Ellouz, 1992). Only34% of decolorization was attained at the end ofthe third cycle, where a progressive darkening ofthe immobilized biomass was observed, probably

due to nucleophilic attack of amino groups of themycelial wall polymers to quinonoid compounds,such as chitin and chitosans (Payne et al., 1992).A dramatic decline in the decolorizing capabilitywas observed during a subsequent fourth cycle(data not shown) indicating that the system’s life-time should be limited to 25 days. Decrease incolor was nearly linear with time, albeit slowerduring the third test. Hence, it appeared thatdecolorization was independent on the concentra-tion of residual color.

3.2. Remo6al of total organic carbon and ofphenolic constituents

The three batches showed an high capability inthe depletion of TOC content. In fact, the rate ofdecrease in TOC concentration was fairly con-stant during the three different experiments. Inthe first batch the maximum TOC removal was73% while in the two subsequent experiments 88and 75% of TOC reduction were achieved (Fig.1). It appeared that the rate of TOC removal wasalso independent on TOC concentrationremaining.

The detoxifying capability was further confi-rmed by the trend of total phenol content. In fact,the removal efficiency of phenols reached themaximum values of 83.5, 88.5 and 78%, respec-tively in the three batches. However, in this case,the total phenol removal was rapid during thefirst 2 days and decreased slowly thereafter. Infact, it is noteworthy to point out the high phenolremoval reached after only 2 days of treatmentwhich ranged from 60 to 80% in the differentbatches. Furthermore an even higher percentageof ortho-diphenols removal was detected andshowed the same trend as total phenols (Fig. 1).

In order to elucidate the detoxifying mecha-nism, the chemical characterization of themonomeric fraction of OMW was performed be-fore and after fungal treatment. The amount ofextractive materials was measured after eachbiodegradation cycle, and the composition of thephenolic monomeric components soluble indichloromethane was estimated by GC-MS spec-troscopy. The amount of extractives was found tobe heavily reduced in all the three batches starting

Table 1Composition and features of the untreated olive-mill wastewa-ter

Parameter Untreated olive-millwastewater

pH 5.30Total organic carbon 15.0Total phenols (as caffeic 1.53

acid)0.32Ortho-diphenols (as caffeic

acid)Total sugars 0.69

0.040Total nitrogen0.16Ca2+

Na+ 0.48K+ 0.34Fe2+ 0.032

All concentrations are expressed in g dm−3.

A. D ’Annibale et al. / Journal of Biotechnology 61 (1998) 209–218 213

Fig. 1. Changes in colour ( ), total organic carbon (�), total phenols () and ortho-diphenols (�) content during three consecutivetreatment cycles of olive mill wastewater by immobilized L. edodes. The effluent was supplemented with saccharose (10 g l−1), yeastextract (1 g l−1) and Tween 80 (0.05%).

from the value of 3.17 g l−1 in the untreatedOMW to 0.70, 0.75 and 0.78 g l−1 at the end ofeach batch respectively. A significant fraction ofthe monomeric components of such extracts wasfound to be composed by phenols (Fig. 2), such ashydroxybenzoic and hydroxyphenylacetic acids,tyrosol and hydroxytyrosol. The phenolic com-pound more recalcitrant to the fungal degradationwas 3-methoxyphenol 1, that was still present atthe end of the second batch and was completelydepleted only at the end of the third cycle. All thediphenolic compounds identified in the OMWextracts, such as catecol 2, 3,4-dihydroxytyrosol 3,and 3,4-dihydroxyphenyl acetic acid 4 were foundto be completely degraded at the end of eachbatch, while the phenolic compounds 5–11 were

found to be completely degraded only at the endof the second and third batch. It is evident thatthe biodegradation proceeds more readily on o-diphenolic structures than on mono-phenols. Infact, the degradation of compounds 5–11 startedonly from the second cycle, possibly in connectionwith the production of fungal oxidative mediatorsduring the secondary phase of fungal growth(Khindaria et al., 1994).

The analysis of the molecular weight pattern ofthe polymeric fraction showed the presence of aconsistent peak with apparent Mr higher than 30kDa, as shown in Fig. 3. This fraction did notappear to be remarkably affected at the end of thefirst cycle of treatment, whereas an evident de-polymerization was attained at the end of thesuccessive cycles.

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3.3. Production of extracellular enzymes and oflow molecular weight agents potentially in6ol6edin the degradation of aromatics

Some enzymatic activities involved in the me-tabolization of aromatic lignin-related compoundsand including laccase (E.C. 1.10.3.2), Mn-depen-dent peroxidase (E.C. 1.11.7.13) were tested dur-ing OMW treatment in the attempt to find anyeventual correlation or to attribute any definedeffect to a particular enzyme. Laccase was appar-ent after three days from OMW addition and didnot appear to attain its maximal activity in theselected cycle period, whereas Mn-dependent per-oxidase activity was detected starting from thesecond cycle and reaching its maximum 6 daysafter the beginning of each cycle (Fig. 4).

During the consecutive cycles of OMW biocon-version the production of extracellular hydrogenperoxide and of oxalic acid was detected, asshown in Fig. 4. In particular, the production of

H2O2 was observed in the first days of the secondand third cycle, reaching its maximal concentra-tion on day 8 and 6, respectively. Oxalic acidproduction appeared to parallel Mn-dependentperoxidase activity.

4. Discussion

L. edodes, strain SC-495, was selected in thisstudy on the basis of its previously reportedOMW-decolorizing ability under non-optimizedconditions (Vinciguerra et al., 1995). In addition,incubation of monomeric, dimeric and polymericaromatics with crude or purified oxidases fromthis fungus resulted in the formation of severalmetabolites, including aromatic ring cleavageproducts (Crestini and Giovannozzi Sermanni,1995; Crestini et al., 1996b; D’Annibale et al.,1996b).

Reduction in color and aromatic compoundscombined with changes in the molecular size dis-tribution of the water-soluble polymeric fractionhave been observed in this study. In particular,depolymerization of the high molecular weightfraction was observed at the end of the secondand third cycle, where the simultaneous produc-tion of laccase and manganese-peroxidase oc-curred. No evident correlations were foundbetween OMW decolorization and laccase andMn peroxidase production by L. edodes. The evi-dent depolymerization attained at the end of thethird cycle was not combined with a high degreeof effluent decolorization.

It is noteworthy that oxalic acid productionparalleled MnP activity, as reported in anotherstudy carried out with nitrogen-limited cultures ofPhanerochaete chrysosporium (Kuan and Tien,1993). Oxalate has been found to accumulate inliquid culture media of other white-rot species,(Dutton et al., 1993) during the secondary phaseof growth (Espejo and Agosin, 1991). This or-ganic acid can play multiple roles in lignin-de-grading fungi. In fact, it can act as an organicchelator, thereby stabilizing the enzymaticallygenerated Mn (III) ions (Waarishi et al., 1992)and accelerating the catalytic turnover of MnP(Kuan and Tien, 1993). Several investigations car-

Fig. 2. Monomeric aromatic compounds extracted from OMWbefore treatment with immobilized mycelium of Lentinulaedodes. (1) 3-methoxyphenol; (2) catechol; (3) 3,4-dihydroxy-phenylethanol; (4) 3,4-dihydroxyphenylacetic acid; (5) 3-hy-droxybenzoic acid; (6) 4-hydroxyphenylethanol; (7) 3-hydrox-yphenylacetic acid; (8) 4-hydroxyphenylacetic acid; (9)3-hydroxyphenylpropionic acid; (10) p-coumaric acid; (11) 3-hydroxybenzoic acid.

A. D ’Annibale et al. / Journal of Biotechnology 61 (1998) 209–218 215

Fig. 3. Size-exclusion-HPLC chromatograms of water-soluble polymeric fraction monitored at 278 nm of control and biotreatedsamples of olive mill wastewater. The column was calibrated with the following polyethyleneglycol (PEG) standards: PEG 30000(retention time 5.2 min); PEG 7500 (retention time 6.7 min); PEG 1000 (retention time 8.4 min); veratryl alcohol (retention time 12.3min).

ried out on the white-rot fungus P. chrysosporiumhave suggested its direct involvement in lignindepolymerization (Akamatsu et al., 1990; Popp etal., 1990). Furthermore, it has been demonstratedthat oxalic acid can reduce Fe(III) to Fe(II) withthe organic acid being simultaneously oxidized tocarbon dioxide (Schmidt et al. 1981; Khindaria etal., 1994). It should be taken into account that theeffluent contained significant amounts of iron(0.03 g dm−3), which, along with hydrogen perox-ide has been shown to generate hydroxyl radicalsin the so-called Fenton’s reactions (Evans et al.,1993). Consequently, the involvement of the Fen-ton’s reaction, which gives rise to the formation ofreactive oxygen species, such as hydroxyl radicalsand superoxide anions, could not be excluded,since all the components required (iron, hydrogenperoxide and oxalate) were present in the reaction

medium. Attempts to find the agents responsiblefor hydrogen peroxide generation were unsuccess-ful. It could be speculated that MnP could beresponsible for H2O2 production. In fact, thisenzyme can produce hydrogen peroxide via twodifferent mechanisms, the first involving the oxi-dation of NAD(P)H (Gold and Glenn, 1988),which can be released in the medium upon cellautolysis. The second mechanism postulates thatMnP can decarboxylate oxalate giving rise to theformation of carbon dioxide and the anion radicalformiate (Khindaria et al., 1994). The latter canreduce O2 to superoxide anion which, in turn, canspontaneously disproportionate to O2 and hydro-gen peroxide. Further investigations are necessaryin order to find the mechanism responsible forhydrogen peroxide formation by the fungus understudy.

A. D ’Annibale et al. / Journal of Biotechnology 61 (1998) 209–218216

Fig. 4. Time course of extracellular laccase ( ), manganese-peroxidase (�), hydrogen peroxide () and oxalic acid (�) producedduring three consecutive treatment cycles of olive mill wastewater by imobilized mycelium of L. edodes. The activities are expressedin mmol min−1 ml−1 and error bars represent the S.D. of data.

5. Conclusions

The OMW treatment with polyurethane-immo-bilized L. edodes resulted in an overall abatementof phenolic and aromatic components as well asin an extensive decolorization and in an apparentdepolymerization of the high molecular weightfraction. Such depolymerization was evident atthe end of the second and third cycle, when thesimultaneous presence of laccase and MnP wasapparent. The best biodegradative results wereobtained in the second batch, when the switch tosecondary metabolism appeared to occur, asshown by the presence of oxalic acid, a typicalsecondary metabolite in white-rot fungi. The dra-matic decline in the decolorizing ability of the

immobilized biomass after the end of the thirdcycle indicated that the limit of the system’s life-time should be 25 days. Nevertheless, removal ofphenols was similar in all three tests indicatingthat the biodegradative metabolism of these com-ponents was not tightly connected with colorremoval.

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