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The average annual global production of olive oil is about 1.6 million tons. In the extraction process, the following two by-products are obtained along with the oil (which accounts for 20% of the total): a solid residue (30% of the total) and a black wastewater (50% of the total) called olive oil mill wastewater (OMW). Most of the solid residue is used as fuel, but the OMW is an environmental problem for the Mediterranean countries. In the 1980s, the Spanish government prohibited the tipping of OMW into rivers. OMW is currently concentrated by evaporation in aerated lagoons, which leaves a black, foul-smelling sludge which is difficult to dispose of. It consists mainly of water (80%) and contains between 4 and 16% organic matter and 2% minerals. Polymeric phenolic compounds similar in structure to lignin give the sludge its characteristic recalcitrant brownish black color (17, 22, 25). Monomeric phenolic compounds which are both antimicrobial and phytotoxic are also present (4, 11, 13, 19, 23). An important step in the degradation of OMW is the breakdown of colored polymeric phenolics (decolorization) to monomers which can subsequently be mineralized (27, 32). Decolorization by Phanerochaete chrysosporium was first reported by Pérez et al. (17). Later, several authors described decolorization of OMW by different white rot fungi (10, 12, 27, 28, 32). It has been shown that there is a significant correlation between OMW decolorization and reduction of total organic carbon and phenolic compounds (3, 14, 29, 32). In research on the ligninolytic enzymes implicated in this degradation process, workers have frequently used chemically defined liquid media (10, 29). Other workers have demonstrated that the enzymes produced in defined lignin-free media are different from those produced in the presence of lignin and lignin-related residues (5, 21). The objective of the research reported here was to determine the pattern of ligninolytic enzymes present in the extracellular fluids of Phanerochaete flavido-alba-decolorized OMW-containing cultures and to compare these enzymes to the enzymes produced in chemically defined residue-free liquid cultures. P. flavido-alba produces lignin peroxidase (LiP), manganese-dependent peroxidase (MnP), and laccase and is able to degrade synthetic lignins, decolorize paper mill wastewaters, and degrade OMW components (9, 10, 18, 20).

Twenty-eight 1-liter Erlenmeyer flasks containing 70 ml of glucose-nitrogen-limited medium (2) supplemented with 40 ppm of Mn(II) were inoculated with P. flavido-alba (18). This medium was shown to be the most suitable medium for OMW decolorization in a previous study (10). We added vacuum-concentrated OMW to one-half of the cultures on day 5. This gave a final concentration of colored material identical to the concentration in the original residue, as estimated by absorbance at 465 nm (20). The remaining flasks, without added OMW, were maintained under identical conditions as controls. Two additional flasks containing OMW were prepared in the same manner but were not inoculated with P. flavido-alba. These flasks served as color controls. The physical and chemical characteristics of the OMW were as follows: pH, 4.7; conductivity, 3,500 µ¹; chemical oxygen demand, 35.5 g liter¹; biological oxygen demand, 25.5 g liter¹; total phenolic compound concentration, 2.8 g liter¹; and ammonia concentration, 25 ppm. The flasks were incubated at 30°C under static conditions, and they were flushed daily starting on day 3 with pure O₂ (3 liters/min for 1 min). Cultures were harvested on day 13, 3 days following the onset of effluent decolorization, when the cultures were visibly decolored. The color was reduced by about 70% at harvest in P. flavido-alba-inoculated cultures compared with uninoculated controls. This amount of decolorization is comparable to the amount observed in previous experiments (10). P. flavido-alba grew better in the residue-containing cultures than in the residue-free controls, as assessed by mycelial dry weight at harvest; the average yields ± standard deviations for six flasks were 274.0 ± 36.0 mg/flask for the OMW-containing flasks and 147.0 ± 16.0 mg/flask for the residue-free controls. The final pHs were not significantly altered in either group of flasks (the pH values were 4.4 to 4.6), indicating that decolorization is not due to artifactual pH changes.

Laccase, MnP, and LiP activities were determined as previously described (15, 16, 31). Table 1 shows the enzymatic activities detected at different steps of the semipurification process. In extracellular fluids, the MnP activity was around eightfold lower in the OMW-containing cultures than in the controls. LiP was not detected in any of the samples tested, and laccase was detected only in OMW-containing cultures. Subsequently, extracellular fluids were concentrated 100-fold by ultrafiltration (Centricon-10; 10,000-M_r cutoff) and were dialyzed against 10 mM sodium acetate (pH 6.0) (Minitan system [Millipore]; 10,000-M_r cutoff); this was followed by passage through a QMA anion-exchange column (Accell; Waters) and further concentration by using Centricon-10. The final concentration achieved by this process was 1,500-fold.

We performed 0.1% sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (8), and the gels were stained with Coomassie blue. Several prominent bands around an apparent molecular weight of 45,000 were visible in 100-fold-concentrated effluent-free culture samples (Fig. 1, lane 4) but not in 100-fold-concentrated OMW-containing culture samples (probably due to masking by residual colored compounds) (Fig. 1, lane 3). These 45,000-M_r bands are typical of MnPs and LiPs found in P. flavido-alba and other white rot fungi (1, 7).

View larger version (105K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of extracellular fluids from P. flavido-alba cultures semipurified by anion-exchange chromatography (Accell column). Lane 1, OMW-containing cultures; lane 2, control cultures; lanes 3 and 4, fluids concentrated 100-fold by ultrafiltration from OMW-containing (lane 3) and control (lane 4) cultures; lane 5, molecular mass markers. Molecular masses (in kilodaltons) are indicated on the right. This gel is representative of the results of three trials.

SDS-PAGE of 1,500-fold-concentrated samples (semipurified by anion exchange) which contained smaller amounts of residual colored compounds resulted in the production of different protein profiles by OMW-containing and control samples (Fig. 1, lanes 1 and 2). In control samples prominent bands were visible at M_rs of 38,000 to 45,000, whereas in the OMW-containing samples bands were observed at molecular weights of 44,000 to 48,000.

Analytical isoelectrofocusing (IEF) (18) of anion-exchange-semipurified control culture samples revealed proteins with isoelectric points (pIs) ranging from 5.7 to 4.5 and an additional band at pI 3.55 (Fig. 2C, lane 2). However, proteins concentrated from OMW-containing cultures had pIs lower than 4.7 (Fig. 2C, lane 3). MnP activity staining (26) of IEF gels revealed that MnPs from control samples had more basic pIs than MnPs from OMW-containing samples (Fig. 2B, lanes 2 and 3). These results strongly suggest that the MnP isoforms present in the decolorized samples were different from those present in control supernatants. A band with MnP activity was also detected in both of the samples with an unusually low pI (less than 3.55) (Fig. 2B, lanes 2 and 3).

View larger version (61K): [in this window] [in a new window]   FIG. 2.   IEF of anion-exchange (Accell column)-semipurified extracellular culture fluids. (A) Gel stained for laccase activity. Lane 1, control culture samples; lane 2, boiled OMW-containing culture samples; lane 3, OMW-containing culture samples; lane 4, P. flavido-alba purified laccase. (B) Gel stained for MnP activity. Lane 1, semipurified P. flavido-alba MnPs; lane 2, control culture samples; lane 3, OMW-containing culture samples. (C) Gel stained for proteins. Lane 1, pI standards; lane 2, control culture samples; lane 3, OMW-containing culture samples. The gels are representative of the results of at least three trials.

A band at an apparent molecular weight of 52,000 was clearly detected in the 1,500-fold-concentrated supernatants from both OMW-containing and control samples (Fig. 1). This band does not correspond to any of the previously described P. flavido-alba ligninolytic enzymes. Two other unidentified bands at M_rs of 66,000 and 80,000 were detected in control samples but not in OMW-containing samples (Fig. 1).

In the presence of OMW, an additional protein with a molecular weight of 94,000 was detected. Presnell et al. (21) also showed that P. chrysosporium is able to produce additional proteins in bleach plant effluent-containing cultures compared to controls (as detected by a comparison of extracellular protein profiles).

Accell-semipurified concentrated samples were loaded onto a Mono Q anion-exchange column fast protein liquid chromatography (FPLC) system. The enzymes were eluted as described by Pérez et al. (18). The FPLC profiles for MnPs were drastically different for control and OMW-containing samples, as expected from the IEF results. Peaks corresponding to MnP isoenzymes eluted at higher salt concentrations in OMW-containing samples than in controls (Fig. 3). These results agreed with those obtained with MnP activity-stained gels. MnP activity was detected in proteins with lower pI values (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   FPLC anion-exchange chromatography of the extracellular fluids from P. flavido-alba control cultures (A) and OMW-containing cultures (B). Dashed line, laccase activity; solid line, MnP activity.

Even though LiP activity was not detected in unconcentrated extracellular fluids from OMW-containing cultures or controls (Table 1), traces of activity were detected in fractions 61 to 64 eluted from the Mono Q column with both samples. The predominant P. flavido-alba LiP has a molecular weight of about 40,000 (unpublished data). No proteins with M_rs around this value were detected in OMW-containing samples. We could not conclude that LiP was not present in our cultures, but if it was present, it must have been present at very low levels.

The laccase activity in OMW-containing samples was resolved as two different peaks by FPLC, which suggests that two different isoenzymes with very similar pIs could have been present (Fig. 3B). No laccase activity was detected in control samples.

Induction of laccase activity in OMW-containing cultures was demonstrated by several pieces of evidence. Laccase activity was not detected in either unconcentrated or semipurified control samples but was detected in both unconcentrated and semipurified OMW-containing media (7.42 and 557 nmol/min · ml) (Table 1). Laccase activity was also detected as a diffuse band (with a pI similar to that of purified P. flavido-alba laccase [18]) in IEF gel analyses of OMW-containing media but not control media (Fig. 2A, lanes 3, 4, and 1). The laccase activity detected was unlikely to be due to oxidation of the test substrate by an inorganic contaminant, as the activity was not detected in boiled samples (Fig. 2A, lane 2).

As shown in Fig. 1, lane 1, a protein with an apparent molecular weight of 94,000 was induced in OMW-containing cultures. Since this molecular weight was very similar to that reported for P. flavido-alba laccase (18), the active fractions from a Mono Q column were analyzed by SDS-PAGE along with the purified laccase. The results shown in Fig. 4 revealed that the two proteins have almost identical molecular weights. Thus, given the similar M_r and pI values of the induced laccase reported here, this enzyme is probably the same P. flavido-alba laccase reported by Pérez et al. (18).

View larger version (65K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE of P. flavido-alba laccase semipurified from OMW-containing samples (lane 1) and P. flavido-alba purified laccase (lane 2).

Several lines of evidence suggest that laccase is involved in OMW biotransformation. First, increases in phenol oxidase activity in the presence of OMW-containing cultures have been reported for two other white rot fungi, Lentinus edodes and Pleurotus ostreatus (12, 32). This induction of laccase may be due to the aromatic compounds present in OMW. Laccase induction by aromatic compounds has been known for many years (6), and such induction has also been demonstrated for P. flavido-alba laccase (24). Second, incubation of raw OMW with phenol oxidase from P. ostreatus resulted in a reduction in the low-M_r phenolic content of up to 90% (12). Third, laccase activity has been demonstrated in P. chrysosporium only recently (30), and therefore previous studies have not concentrated on the role of laccase activity (10, 29).

Our results confirm that OMW influences the production of ligninolytic enzymes by P. flavido-alba. MnPs are the predominant enzymes in OMW decolorized by P. flavido-alba, and laccase not only is present but is strongly induced in such supernatant solutions. This report may contribute to a better understanding of the enzymes implicated in OMW decolorization, and the results suggest that laccase and MnP play an important role in this biodegradation process by white rot fungi.