APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010

July 1998, p. 2726–2729 Vol. 64, No. 7

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Phanerochaete flavido-alba Laccase Induction and Modificationof Manganese Peroxidase Isoenzyme Pattern in Decolorized

Olive Oil Mill WastewatersJ. PEREZ, T. DE LA RUBIA, O. BEN HAMMAN, AND J. MARTINEZ*

Departamento de Microbiologıa, Facultad de Farmacia, Campus de Cartuja, Universidad de Granada,18071 Granada, Spain

Received 29 December 1997/Accepted 23 March 1998

Lignin-degrading enzymes were partially purified from supernatant solutions obtained from Phanerochaeteflavido-alba-decolorized olive oil mill wastewaters (OMW). The dominant enzymes, manganese peroxidases,exhibited different isoform patterns in decolorized OMW-containing cultures than in residue-free samples.Laccase induction was also detected in OMW-containing cultures but not in control cultures.

The average annual global production of olive oil is about1.6 million tons. In the extraction process, the following twoby-products are obtained along with the oil (which accounts for20% of the total): a solid residue (30% of the total) and a blackwastewater (50% of the total) called olive oil mill wastewater(OMW). Most of the solid residue is used as fuel, but theOMW is an environmental problem for the Mediterraneancountries. In the 1980s, the Spanish government prohibited thetipping of OMW into rivers. OMW is currently concentratedby evaporation in aerated lagoons, which leaves a black, foul-smelling sludge which is difficult to dispose of. It consistsmainly of water (80%) and contains between 4 and 16% or-ganic matter and 2% minerals. Polymeric phenolic compoundssimilar in structure to lignin give the sludge its characteristicrecalcitrant brownish black color (17, 22, 25). Monomeric phe-nolic compounds which are both antimicrobial and phytotoxicare also present (4, 11, 13, 19, 23). An important step in thedegradation of OMW is the breakdown of colored polymericphenolics (decolorization) to monomers which can subse-quently be mineralized (27, 32). Decolorization by Phanero-chaete chrysosporium was first reported by Perez et al. (17).Later, several authors described decolorization of OMW bydifferent white rot fungi (10, 12, 27, 28, 32). It has been shownthat there is a significant correlation between OMW decolor-ization and reduction of total organic carbon and phenoliccompounds (3, 14, 29, 32). In research on the ligninolyticenzymes implicated in this degradation process, workers havefrequently used chemically defined liquid media (10, 29).Other workers have demonstrated that the enzymes producedin defined lignin-free media are different from those producedin the presence of lignin and lignin-related residues (5, 21).The objective of the research reported here was to determinethe pattern of ligninolytic enzymes present in the extracellularfluids of Phanerochaete flavido-alba-decolorized OMW-con-taining cultures and to compare these enzymes to the enzymesproduced in chemically defined residue-free liquid cultures. P.flavido-alba produces lignin peroxidase (LiP), manganese-de-pendent peroxidase (MnP), and laccase and is able to degradesynthetic lignins, decolorize paper mill wastewaters, and de-grade OMW components (9, 10, 18, 20).

Twenty-eight 1-liter Erlenmeyer flasks containing 70 ml ofglucose-nitrogen-limited medium (2) supplemented with 40ppm of Mn(II) were inoculated with P. flavido-alba (18). Thismedium was shown to be the most suitable medium for OMWdecolorization in a previous study (10). We added vacuum-concentrated OMW to one-half of the cultures on day 5. Thisgave a final concentration of colored material identical to theconcentration in the original residue, as estimated by absor-bance at 465 nm (20). The remaining flasks, without addedOMW, were maintained under identical conditions as controls.Two additional flasks containing OMW were prepared in thesame manner but were not inoculated with P. flavido-alba.These flasks served as color controls. The physical and chem-ical characteristics of the OMW were as follows: pH, 4.7;conductivity, 3,500 mV21; chemical oxygen demand, 35.5 gliter21; biological oxygen demand, 25.5 g liter21; total phenoliccompound concentration, 2.8 g liter21; and ammonia concen-tration, 25 ppm. The flasks were incubated at 30°C under staticconditions, and they were flushed daily starting on day 3 withpure O2 (3 liters/min for 1 min). Cultures were harvested onday 13, 3 days following the onset of effluent decolorization,when the cultures were visibly decolored. The color was re-duced by about 70% at harvest in P. flavido-alba-inoculatedcultures compared with uninoculated controls. This amount ofdecolorization is comparable to the amount observed in pre-vious experiments (10). P. flavido-alba grew better in the res-idue-containing cultures than in the residue-free controls, asassessed by mycelial dry weight at harvest; the average yields 6standard deviations for six flasks were 274.0 6 36.0 mg/flask forthe OMW-containing flasks and 147.0 6 16.0 mg/flask for theresidue-free controls. The final pHs were not significantly al-tered in either group of flasks (the pH values were 4.4 to 4.6),indicating that decolorization is not due to artifactual pHchanges.

Laccase, MnP, and LiP activities were determined as previ-ously described (15, 16, 31). Table 1 shows the enzymaticactivities detected at different steps of the semipurificationprocess. In extracellular fluids, the MnP activity was aroundeightfold lower in the OMW-containing cultures than in thecontrols. LiP was not detected in any of the samples tested, andlaccase was detected only in OMW-containing cultures. Sub-sequently, extracellular fluids were concentrated 100-fold byultrafiltration (Centricon-10; 10,000-Mr cutoff) and were dia-lyzed against 10 mM sodium acetate (pH 6.0) (Minitan system[Millipore]; 10,000-Mr cutoff); this was followed by passage

* Corresponding author. Mailing address: Departamento de Micro-biologıa, Facultad de Farmacia, Campus de Cartuja, Universidad deGranada, 18071 Granada, Spain. Phone: 34 58 243873. Fax: 34 58246235. E-mail: Jmtnez@platon.ugr.es.

2726

on October 29, 2018 by guest

http://aem.asm

.org/D

ownloaded from

through a QMA anion-exchange column (Accell; Waters) andfurther concentration by using Centricon-10. The final concen-tration achieved by this process was 1,500-fold.

We performed 0.1% sodium dodecyl sulfate–10% polyacryl-amide gel electrophoresis (SDS-PAGE) as described byLaemmli (8), and the gels were stained with Coomassie blue.Several prominent bands around an apparent molecular weightof 45,000 were visible in 100-fold-concentrated effluent-freeculture samples (Fig. 1, lane 4) but not in 100-fold-concentrat-ed OMW-containing culture samples (probably due to maskingby residual colored compounds) (Fig. 1, lane 3). These 45,000-Mr bands are typical of MnPs and LiPs found in P. flavido-albaand other white rot fungi (1, 7).

SDS-PAGE of 1,500-fold-concentrated samples (semipuri-fied by anion exchange) which contained smaller amounts ofresidual colored compounds resulted in the production of dif-ferent protein profiles by OMW-containing and control sam-ples (Fig. 1, lanes 1 and 2). In control samples prominent bandswere visible at Mrs of 38,000 to 45,000, whereas in the OMW-containing samples bands were observed at molecular weightsof 44,000 to 48,000.

Analytical isoelectrofocusing (IEF) (18) of anion-exchange-semipurified control culture samples revealed proteins withisoelectric points (pIs) ranging from 5.7 to 4.5 and an addi-tional band at pI 3.55 (Fig. 2C, lane 2). However, proteinsconcentrated from OMW-containing cultures had pIs lowerthan 4.7 (Fig. 2C, lane 3). MnP activity staining (26) of IEF gelsrevealed that MnPs from control samples had more basic pIs

than MnPs from OMW-containing samples (Fig. 2B, lanes 2and 3). These results strongly suggest that the MnP isoformspresent in the decolorized samples were different from thosepresent in control supernatants. A band with MnP activity wasalso detected in both of the samples with an unusually low pI(less than 3.55) (Fig. 2B, lanes 2 and 3).

A band at an apparent molecular weight of 52,000 wasclearly detected in the 1,500-fold-concentrated supernatantsfrom both OMW-containing and control samples (Fig. 1). Thisband does not correspond to any of the previously describedP. flavido-alba ligninolytic enzymes. Two other unidentified bandsat Mrs of 66,000 and 80,000 were detected in control samplesbut not in OMW-containing samples (Fig. 1).

In the presence of OMW, an additional protein with a mo-lecular weight of 94,000 was detected. Presnell et al. (21) alsoshowed that P. chrysosporium is able to produce additional pro-teins in bleach plant effluent-containing cultures compared tocontrols (as detected by a comparison of extracellular proteinprofiles).

Accell-semipurified concentrated samples were loaded ontoa Mono Q anion-exchange column fast protein liquid chroma-tography (FPLC) system. The enzymes were eluted as de-scribed by Perez et al. (18). The FPLC profiles for MnPs weredrastically different for control and OMW-containing samples,as expected from the IEF results. Peaks corresponding to MnPisoenzymes eluted at higher salt concentrations in OMW-con-taining samples than in controls (Fig. 3). These results agreedwith those obtained with MnP activity-stained gels. MnP activ-ity was detected in proteins with lower pI values (Fig. 2B).

Even though LiP activity was not detected in unconcentratedextracellular fluids from OMW-containing cultures or controls(Table 1), traces of activity were detected in fractions 61 to 64

FIG. 1. SDS-PAGE of extracellular fluids from P. flavido-alba cultures semi-purified by anion-exchange chromatography (Accell column). Lane 1, OMW-containing cultures; lane 2, control cultures; lanes 3 and 4, fluids concentrated100-fold by ultrafiltration from OMW-containing (lane 3) and control (lane 4)cultures; lane 5, molecular mass markers. Molecular masses (in kilodaltons) areindicated on the right. This gel is representative of the results of three trials.

FIG. 2. IEF of anion-exchange (Accell column)-semipurified extracellularculture fluids. (A) Gel stained for laccase activity. Lane 1, control culture sam-ples; lane 2, boiled OMW-containing culture samples; lane 3, OMW-containingculture samples; lane 4, P. flavido-alba purified laccase. (B) Gel stained for MnPactivity. Lane 1, semipurified P. flavido-alba MnPs; lane 2, control culture sam-ples; lane 3, OMW-containing culture samples. (C) Gel stained for proteins.Lane 1, pI standards; lane 2, control culture samples; lane 3, OMW-containingculture samples. The gels are representative of the results of at least three trials.

TABLE 1. Purification steps and enzymatic activities in P. flavido-alba extracellular fluids

Purification step Concn (fold)

Enzyme activities (nmol/min z ml) ina:

Control cultures OMW-containing cultures

MnP Laccase LiP MnP Laccase LiP

Extracellular fluids 1 340 NDb ND 41.7 7.42 NDAccell column concentration and dialysis 1,500 23,760 ND ND 1,900 557 ND

a Means determined by using the values from three flasks (extracellular fluids) or from three repetitions with pooled samples (Accell column concentration anddialysis). The standard deviations were less than 10% of the averages.

b ND, not detected.

VOL. 64, 1998 LIGNINOLYTIC ENZYMES IN DECOLORIZED OLIVE OIL WASTES 2727

on October 29, 2018 by guest

http://aem.asm

.org/D

ownloaded from

eluted from the Mono Q column with both samples. The pre-dominant P. flavido-alba LiP has a molecular weight of about40,000 (unpublished data). No proteins with Mrs around thisvalue were detected in OMW-containing samples. We couldnot conclude that LiP was not present in our cultures, but if itwas present, it must have been present at very low levels.

The laccase activity in OMW-containing samples was re-solved as two different peaks by FPLC, which suggests that twodifferent isoenzymes with very similar pIs could have beenpresent (Fig. 3B). No laccase activity was detected in controlsamples.

Induction of laccase activity in OMW-containing cultureswas demonstrated by several pieces of evidence. Laccase ac-tivity was not detected in either unconcentrated or semipuri-fied control samples but was detected in both unconcentratedand semipurified OMW-containing media (7.42 and 557 nmol/min z ml) (Table 1). Laccase activity was also detected as adiffuse band (with a pI similar to that of purified P. flavido-albalaccase [18]) in IEF gel analyses of OMW-containing mediabut not control media (Fig. 2A, lanes 3, 4, and 1). The laccaseactivity detected was unlikely to be due to oxidation of the testsubstrate by an inorganic contaminant, as the activity was notdetected in boiled samples (Fig. 2A, lane 2).

As shown in Fig. 1, lane 1, a protein with an apparentmolecular weight of 94,000 was induced in OMW-containingcultures. Since this molecular weight was very similar to thatreported for P. flavido-alba laccase (18), the active fractionsfrom a Mono Q column were analyzed by SDS-PAGE alongwith the purified laccase. The results shown in Fig. 4 revealed

that the two proteins have almost identical molecular weights.Thus, given the similar Mr and pI values of the induced laccasereported here, this enzyme is probably the same P. flavido-albalaccase reported by Perez et al. (18).

Several lines of evidence suggest that laccase is involved inOMW biotransformation. First, increases in phenol oxidaseactivity in the presence of OMW-containing cultures have beenreported for two other white rot fungi, Lentinus edodes andPleurotus ostreatus (12, 32). This induction of laccase may bedue to the aromatic compounds present in OMW. Laccaseinduction by aromatic compounds has been known for manyyears (6), and such induction has also been demonstrated forP. flavido-alba laccase (24). Second, incubation of raw OMWwith phenol oxidase from P. ostreatus resulted in a reduction inthe low-Mr phenolic content of up to 90% (12). Third, laccaseactivity has been demonstrated in P. chrysosporium only re-cently (30), and therefore previous studies have not concen-trated on the role of laccase activity (10, 29).

Our results confirm that OMW influences the production ofligninolytic enzymes by P. flavido-alba. MnPs are the predom-inant enzymes in OMW decolorized by P. flavido-alba, andlaccase not only is present but is strongly induced in suchsupernatant solutions. This report may contribute to a betterunderstanding of the enzymes implicated in OMW decoloriza-tion, and the results suggest that laccase and MnP play animportant role in this biodegradation process by white rotfungi.

We are very grateful to the Spanish CICYT for financial support(project BIO96-0393). J.P. was supported by grants from the Univer-sity of Granada and the Spanish Education Science Ministry.

REFERENCES1. Ben Hamman, O., T. de la Rubia, and J. Martınez. 1997. Effect of carbon

and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba. J. Appl. Microbiol. 83:751–757.

2. Bonnarme, P., and T. W. Jeffries. 1990. Mn(II) regulation of lignin peroxi-dases and manganese peroxidases from lignin-degrading white rot fungi.Appl. Environ. Microbiol. 56:210–217.

3. Boominathan, K., T. M. D’ Souza, P. S. Naidu, C. G. Dosoretz, and C. A.Reddy. 1993. Temporal expression of the major lignin peroxidase genes ofPhanerochaete chrysosporium. Appl. Environ. Microbiol. 59:3946–3950.

4. Capasso, R., G. Cristinzio, A. Evidente, and F. Scognamiglio. 1992. Isolation,spectroscopy and selective phytotoxic effects of polyphenols from vegetablewaste waters. Phytochemistry 31:4125–4128.

5. Datta, A., A. Betterman, and T. K. Kirk. 1991. Identification of a specificmanganese peroxidase among ligninolytic enzymes secreted by Phanero-chaete chrysosporium during wood decay. Appl. Environ. Microbiol. 57:1453–1460.

6. Fahraeus, G., V. Tullander, and H. Ljunggren. 1958. Production of high

FIG. 3. FPLC anion-exchange chromatography of the extracellular fluidsfrom P. flavido-alba control cultures (A) and OMW-containing cultures (B).Dashed line, laccase activity; solid line, MnP activity.

FIG. 4. SDS-PAGE of P. flavido-alba laccase semipurified from OMW-con-taining samples (lane 1) and P. flavido-alba purified laccase (lane 2).

2728 PEREZ ET AL. APPL. ENVIRON. MICROBIOL.

on October 29, 2018 by guest

http://aem.asm

.org/D

ownloaded from

laccase yields in cultures of fungi. Physiol. Plant. 11:631–643.7. Hattaka, A. 1994. Lignin degrading enzymes from selected white-rot fungi.

Production and role in lignin degradation. FEMS Microbiol. Rev. 13:125–135.

8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

9. Madrid, F., T. de la Rubia, and J. Martınez. 1995. Effect of Phanerochaeteflavido-alba on aromatic acids present in olive oil mill waste waters. Toxicol.Environ. Chem. 51:161–168.

10. Martınez, J., O. Ben Hamman, and T. de la Rubia. 1997. Decoloracion dealpechın por Phanerochaete flavido-alba, abstr. 342, p. 155. In Abstracts ofXVI Congreso de la SEM 1997. Sociedad Espanola de Microbiologıa, Bar-celona, Spain.

11. Martınez, J., J. Perez, E. Moreno, and A. Ramos-Cormenzana. 1986. Inci-dencia del efecto antimicrobiano del alpechın en su posible aprovecha-miento. Grasas Aceites 37:215–223.

12. Martiriani, L., P. Giardina, L. Marzullo, and G. Sannia. 1996. Reduction ofphenol content and toxicity in olive oil mill waste waters with the ligninolyticfungus Pleurotus ostreatus. Water Res. 30:1914–1918.

13. Moreno, E., J. Perez, A. Ramos-Cormenzana, and J. Martınez. 1987. Anti-microbial effect of waste water from olive oil extraction plants selecting soilbacteria after incubation with diluted waste. Microbios 51:169–174.

14. Morrison, W. H., and M. M. Mulder. 1994. Pyrolysis mass spectrometry andpyrolysis gas chromatography-mass spectrometry of ester- and ether-linkedphenolic acids in coastal Bermudagrass cell walls. Phytochemistry 35:1143–1151.

15. Niku-Paavola, M.-L., E. Karhunen, P. Salola, and V. Raunio. 1988. Ligni-nolytic enzymes of the white rot fungus Phlebia radiata. Biochem. J. 254:877–884.

16. Paszczynski, A., V. B. Huynh, and R. Crawford. 1985. Enzymatic activities ofan extracellular manganese dependent peroxidase from Phanerochaetechrysosporium: FEMS Microbiol. Lett. 29:37–41.

17. Perez, J., M. T. Hernandez, A. Ramos-Cormenzana, and J. Martınez. 1987.Caracterizacon de fenoles del pigmento del alpechın y transformacion porPhanerochaete chrysosporium. Grasas Aceites 38:367–371.

18. Perez, J., J. Martınez, and T. de la Rubia. 1996. Purification and partialcharacterization of a laccase from the white rot fungus Phanerochaete fla-vido-alba. Appl. Environ. Microbiol. 62:4263–4267.

19. Perez, J., T. de la Rubia, J. Moreno, and J. Martınez. 1992. Phenolic contentand antibacterial activity of olive oil waste waters. Environ. Toxicol. Chem.11:489–495.

20. Perez, J., L. Saez, T. de la Rubia, and J. Martınez. 1997. Phanerochaete

flavido-alba ligninolytic activities and decolorization of partially bio-depu-rated paper mill wastes. Water Res. 31:495–502.

21. Presnell, T. L., H. Fukui, T. W. Joyce, and H.-M. Chang. 1992. Bleach planteffluent influences enzyme production by Phanerochaete chrysosporium. En-zyme Microb. Technol. 14:184–189.

22. Ragazzi, E., E. Veronesse, and A. Pietrogrande. 1967. Ricerche sui constitu-enti idrosolubili delle olive. II. Pigmenti e polisacaridi. Ann. Chim. 57:1398–1413.

23. Rodrıguez, M. M., J. Perez, A. Ramos-Cormenzana, and J. Martınez. 1988.Effect of extracts obtained from olive oil mill waste waters on Bacillusmegaterium ATCC 33085. J. Appl. Bacteriol. 64:219–226.

24. Ruiz, E., T. de la Rubia, and J. Martınez. 1997. Efecto del cobre y com-puestos aromaticos sobre la actividad lacasa de Phanerochaete flavido-alba,abstr. 114, p. 76. In Abstracts of XVI Congreso de la SEM 1997. SociedadEspanola de Microbiologıa, Barcelona, Spain.

25. Sainz-Jimenez, C., G. Gomez Alarcon, and J. W. de Leeuw. 1986. Chemicalproperties of the polymer isolated in fresh vegetation water and sludgeevaporation ponds, p. 41–60. In Food and Agriculture Organization (ed.),International Symposium on Olive By Products Valorization 1986. Publica-tion Division of the United Nations Food and Agriculture Organization,Madrid, Spain.

26. Sarkanen, S., R. A. Razal, T. Piccariello, E. Yamamoto, and N. G. Lewis.1991. Lignin peroxidase: toward a clarification of its role in vivo. J. Biol.Chem. 266:3636–3643.

27. Sayadi, S., and R. Ellouz. 1992. Decolourization of olive oil mill waste watersby the white rot fungus Phanerochaete chrysosporium: involvement of thelignin-degrading system. Appl. Microbiol. Biotechnol. 37:813–817.

28. Sayadi, S., and R. Ellouz. 1993. Screening of white rot fungi for the treat-ment of olive mill waste waters. J. Chem. Technol. Biotechnol. 57:141–146.

29. Sayadi, S., and R. Ellouz. 1995. Roles of lignin peroxidase and manganeseperoxidase from Phanerochaete chrysosporium in the decolorization of olivemill wastewaters. Appl. Environ. Microbiol. 61:1098–1103.

30. Srinivasan, C., T. M. D’Souza, K. Boominathan, and C. A. Reddy. 1995.Demonstration of laccase in the white rot basidiomycete Phanerochaetechrysosporium BKM-F1767. Appl. Environ. Microbiol. 61:4274–4277.

31. Tien, M., and T. K. Kirk. 1984. Lignin-degrading enzyme from Phanero-chaete chrysosporium: purification, characterization, and catalytic propertiesof a unique H2O2-requiring oxygenase. Proc. Natl. Acad. Sci. USA 81:2280–2284.

32. Vinciguerra, V., A. D’Annibale, G. Delle Monache, and G. G. Sermanni.1995. Correlated effect during bioconversion of waste olive waters by Lenti-nus edodes. Bioresour. Technol. 51:221–226.

VOL. 64, 1998 LIGNINOLYTIC ENZYMES IN DECOLORIZED OLIVE OIL WASTES 2729

on October 29, 2018 by guest

http://aem.asm

.org/D

ownloaded from