Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate

Soil Biology & Biochemistry 36 (2004) 1545–1551 www.elsevier.com/locate/soilbio

Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate Cˇeneˇk Novotny´a,*, Katerˇina Svobodova´a, Pavla Erbanova´a, Toma´sˇ Cajthamla, Aparna Kasinatha, Elke Langb, Va´clav Sˇasˇeka a

Laboratory of Experimental Mycology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 142 20 Prague 4, Czech Republic b Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b 38124 Braunschweig, Germany Received in revised form 21 April 2004

Abstract Ligninolytic fungi can be used for remediation of pollutants in water and soil. Extracellular peroxidases and laccases have been shown to oxidize recalcitrant compounds in vitro but the likely significance of individual enzyme levels in vivo remains unclear. This study documents the amounts and activities of Mn-dependent peroxidase (MnP), lignin peroxidase and laccase (LAC) in various species of ligninolytic fungi grown in liquid medium and soil and their effect on degradation of polycyclic aromatic hydrocarbons (anthracene and pyrene), a polychlorinated biphenyl mixture (Delor 106) and a number of synthetic dyes. Stationary cultures of a highly degradative strain Irpex lacteus exhibited 380-fold and 2-fold increase in production of MnP and LAC, respectively, compared to submerged cultures. Addition of Tween 80 to the submerged culture increased MnP levels 260-fold. High levels of MnP correlated with efficient decolorization of Reactive Orange 16 azo dye but not of Remazol Brilliant Blue R anthraquinone dye. Degradation of anthracene and pyrene in spiked soil by straw-grown explorative mycelium of Phanerochaete chrysosporium, Trametes versicolor and Pleurotus ostreatus showed the importance of MnP and LAC levels secreted into the soil. The importance of high fungal enzyme levels for efficient degradation of recalcitrant compounds was better demonstrated in liquid media compared to the same strains growing in soil. q 2004 Elsevier Ltd. All rights reserved. Keywords: Ligninolytic fungi; Bioremediation; Organopollutants; Peroxidases; Laccase; Degradation rate

1. Introduction Environmental pollution with hazardous wastes containing recalcitrant xenobiotic chemicals has become one of the major ecological problems. Unlike the naturally occurring organic compounds that are readily degraded upon introduction into the environment, some of these synthetic chemicals are extremely resistant to biodegradation by native microorganisms (Fernando and Aust, 1994). Ligninolytic fungi causing white rot of the wood have been shown to degrade and mineralize a large variety of recalcitrant compounds due to the nonspecificity of their

* Corresponding author. Tel.: C420-2-9644-2357; fax: C420-2-96442384. ˇ . Novotny´). E-mail address: [email protected] (C 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.07.019

enzyme machinery. Many of those compounds are major environmental pollutants such as munitions waste, pesticides, organochlorines, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), synthetic dyes, wood preservatives and synthetic polymers (Pointing, 2001). White rot fungi (such as Phanerochaete chrysosporium, Trametes versicolor, etc.) typically secrete one or more of the three principal ligninolytic enzymes (Hatakka, 1994), i.e. lignin peroxidase (LiP, E.C. 1.11.1.14), Mn-dependent peroxidase (MnP, E.C. 1.11.1.13) and phenol oxidase (laccase) (LAC, E.C. 1.10.3.2) (Thurston, 1994; Orth and Tien, 1995). A number of other enzymes are produced in parallel including other peroxidases, enzymes producing H2O2 required by peroxidases (e.g. glyoxal oxidase and superoxide dismutase), and enzymes linked to lignocellulose degradation pathways (e.g. glucose oxidase and aryl

1546

Cˇ. Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551

alcohol oxidase), whose role in lignin degradation is poorly understood (Pointing, 2001). The physiology of LiP, MnP and LAC has been studied extensively (Hatakka, 1994; Thurston, 1994). These enzymes have been shown to take part in vitro transformation of nonpolymeric, recalcitrant pollutants such as nitrotoluenes (Van Acken et al., 1999), PAHs (Hammel et al., 1991; Johannes et al., 1996), organic and synthetic dyes (Ollikka et al, 1993; Heinfling et al., 1998), and pentachlorophenol (Lin et al., 1990). The importance of high extracellular levels of these enzymes to enable the efficient degradation of recalcitrant compounds under in vivo conditions relates to the sorption and complexing of enzymes in soil and the probable loss of much of their activity once externalized (Stotzky and Burns, 1982). The reason for our poor understanding is the high complexity of the biodegradation mechanisms involved, where in addition to the above ligninolytic enzymes, other biochemical systems and interactions may influence the rate of the bioremediation process, namely cytochrome P450 monooxygenase system (Bezalel et al., 1997), hydroxyl radicals and the level of H2O2 produced by the fungal organism (Kotterman et al., 1996; Tanaka et al., 1999). The limited bioavailability of pollutants results from their sorption to soil particles or a covalent coupling with soil organic matter (Ru¨ttimann-Johnson and Lamar, 1997; Ka¨stner et al., 1999) as well as from the sorption to the fungal mycelium due to their hydrophobic properties (Providenti et al., 1993; Wang and Yu, 1998; Gramss et al., 1999). The research reported here was aimed at evaluating the importance of elevated levels of extracellular, ligninolytic enzyme activities for rapid and efficient degradation of selected recalcitrant compounds under a variety of conditions. Different strains of ligninolytic fungi and several major organopollutants, such as synthetic dyes, PAHs and PCBs were used and their degradation studied in both water and soil.

2. Materials and methods 2.1. Microorganisms Phanerochaete chrysosporium strain ME 446 (ATCC 34541), Trametes versicolor strain CCBAS 614, Coriolopsis polyzona strain CCBAS 740, Pleurotus ostreatus strain 3004 and Irpex lacteus strain 617/93 were obtained from the Culture Collection of Basidiomycetes of the Academy of Sciences, Prague (CCBAS). Fungal cultures were maintained on malt extract/glucose medium agar slants (Novotny´ et al., 2000). 2.2. Growth in liquid media and in soil In all of the experiments the cultures were run in triplicate. The data shown represent the mean values G standard deviation values. The following growth media

were used to cultivate the fungi in liquid media: low nitrogen mineral medium (LNMM) pH 4.5 (Tien and Kirk, 1988), malt extract/glucose medium pH 4.5 (Novotny´ et al., 2000) and malt extract broth medium (20 g lK1; Oxoid, UK). Shallow stationary cultures and submerged cultures were prepared and used as described by Kasinath et al. (2003). The inoculation was from 7-d stationary cultures, preinoculated with two 0.9-cm agar plugs covered with fungal mycelium, gently homogenized in a Waring blender (low speed, three times, 20 s) (Novotny´ et al., 2000). The cultures were spiked with PCBs (0.9 mg lK1) or synthetic dyes (150 mg lK1) and their biodegradation measured, together with the extracellular activities of ligninolytic enzymes and fungal growth (Novotny´ et al., 1997, 2001). Fungal cultures immobilized on polyurethane (PUF) or pinewood (PW) cubes were prepared in two steps (Kasinath et al. 2003). First, solid support consisting of 1-cm3 cubes (3 g dry PUF or 5 g dry PW) was autoclaved in 200 ml of malt extract broth medium (20 g lK1, Oxoid, UK) in 500-ml flasks, inoculated with two 0.9-cm agar plugs covered with fungal mycelium and incubated on a rotary shaker (110 rpm, 25 8C) for 4 days. After the fungus colonized the solid support, the medium was drained and the immobilized fungus aseptically transferred to a reactor vessel. The culture was stabilized by a 5-d circulation of LNMM at 25 8C and used for biodegradation of synthetic dyes (Kasinath et al., 2003). Aerated tube reactors used for cultivation of fungi in soil consisted of compartments of coarse-milled straw and soil separated by a nylon web (Novotny´ et al., 1999). The dry soil was contaminated with anthracene and pyrene (50 mg gK1 each) using sterilized sand spiked with both PAHs. The soil in the reactor was colonized by fungal mycelium growing up from the pre-colonized straw substrate in the former compartment and fungal growth, synthesis of ligninolytic enzymes and removal of PAHs were measured during incubation (Novotny´ et al., 1999). In liquid-medium cultures, fungal growth was estimated gravimetrically as dry biomass obtained by drying washed fungal mycelium at 110 8C overnight. In straw- and soil compartments of the tube reactor cultures, fungal growth was measured using the ergosterol method (Davis and Lamar, 1992). 2.3. Biodegradation and analyses of recalcitrant organopollutants PCBs were added into the liquid medium as a stock solution of a Delor 106 mixture (Chemko, Slovak Republic) in acetone at a final concentration of 0.9 mg lK1. PCBs were extracted with hexane, measured by gas chromatography using electron-capture detector (GC-ECD) and quantitated by application of a sum of characteristic peaks selected from the standard Delor 106 (Novotny´ et al., 1997).

ˇ . Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551 C

Synthetic dyes were used in the liquid medium at a concentration of 150 mg lK1. Their decolorization by fungal cultures was detected spectrophotometrically at the corresponding absorption maxima of the individual dye compounds (Novotny´ et al., 2001; Kasinath et al. 2003). Textile industry coloring bath effluents were 10-times diluted in the growth medium and their decolorization was measured spectrophotometrically at the corresponding absorption maximum. The initial concentration of synthetic dyes in agar plates was 200 mg lK1. A plug cut out of a malt extract/glucose agar culture of the fungus was put in a well (10-mm diameter) cut in the LNMM agar containing a dye and the plate was incubated at 28 8C. Decolorized zone formation was observed at regular time intervals. In order to measure the removal of PAHs the soil was first extracted with hexane/acetone mixture in a Soxhlet apparatus, the extracts were evaporated and the residues dissolved in methanol. The samples were than analyzed using HPLC (Novotny´ et al., 1999). 2.4. Enzyme assays Extracellular ligninolytic enzyme activities were measured in stationary and submerged culture fluids (Novotny´ et al., 2001), in immobilized culture fluids (Kasinath et al., 2003) and in extracts from the straw/soil cultures when the two solid phases (straw and soil colonized by the fungus) were treated separately (Novotny´ et al., 1999). Lyophilized soil and straw from the tube reactor cultures were extracted with 50 mM succinate/lactate buffer pH 4.5 and desalted on a Sephadex G25 column (Pharmacia, Sweden). LiP was assayed with veratryl alcohol as the substrate (Vyas et al., 1994). MnP was determined with 3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone hydrochloride as the chromogen (Vyas et al., 1994). LAC was estimated with 2,2-azinobis-3ethylbenzo-thiazoline-6-sulfonic acid as the substrate (Niku-Paavola et al., 1988). One unit of enzyme activity produced 1 mmol of reaction product minK1 under the assay conditions at room temperature. 2.5. Chemicals All chemicals were of analytical grade. The synthetic dyes (Reactive Orange 16, RO16; Remazol Brilliant Blue R, RBBR; Poly R-478) were purchased from SigmaAldrich, USA. The composition of textile coloring bath liquids obtained from the Jitex Pisek a.s. textile mill is described by Kasinath et al. (2003). Except for the Acid Black bath, other baths were mixtures of different textile dyestuffs (in parentheses): Drimaren Blue (Drimaren Violet, Levafix Royal Blue), Drimaren Red (Drimaren Blue, Drimaren Brilliant Orange, Levafix Brilliant Red), Remazol Green (Yoracron Green, Remazol Brilliant Gel 6). The commercial PCB mixture Delor 106, an

1547

analogue of Arochlor 1260 containing 60% bound chlorine, was a product of former Chemko Stra´zˇske´ (Slovak Republic). Anthracene and pyrene were obtained from Sigma-Aldrich, USA and Fluka, Switzerland, respectively; all PAH standards were purchased from Sigma-Aldrich, USA.

3. Results The biodegradation of recalcitrant pollutants by ligninolytic fungal enzymes in vitro has been documented (Pointing, 2001). However, under in vivo conditions, other biochemical systems, processes and interactions can either contribute to degradation of the pollutant (e.g. fungal cytochrome P450 monooxygenase system, hydroxyl radical formation by the fungus) or limit the degradation rate (e.g. low bioavailability of the pollutant due to sorption to soil particles, hydrophobicity of the pollutant molecule). Our purpose was to provide information about the degradation rates and enzyme levels synthesized by degrading fungal cultures. In order to document this relationship, a broad spectrum of recalcitrant pollutants of different chemical structures was used. Such a correlation would have a significant practical importance since it would show whether it is worth selecting for strains with high ligninolytic enzyme activities and/or whether, when developing the bioremediation technology, we should focus on the conditions promoting maximal enzyme synthesis and secretion. As the synthesis of ligninolytic enzymes depends on cultivation conditions (Kirk and Farrell, 1987), the significance of high levels of ligninolytic enzymes was assessed in various types of fungal cultures.

3.1. Liquid agitated and nonagitated fungal cultures Agitated and nonagitated cultures of I. lacteus, a fungus producing all three major ligninolytic enzymes (Novotny´ et al., 2000), were compared with respect to extracellular enzyme synthesis and the capability of decolorizing the azo dye RO16 and the anthraquinone dye RBBR (Table 1). In submerged culture, the production of MnP, LiP and LAC were significantly reduced in comparison with stationary culture although growth yields were similar (1.5G0.3 and 1.6G0.2 g dry biomass lK1, respectively). The difference in enzyme activities correlated with a lower rate of decolorization of RO16, but not of RBBR. The addition of Tween detergent in the growth medium, shown previously to increase the production of extracellular ligninolytic enzymes in submerged cultures (Leisola and Fiechter, 1985), increased the production of extracellular MnP but not LiP and LAC (Table 1). As a result, the rate of decolorization of RO16 increased to reach a similar low

1548

Cˇ. Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551

Table 1 Effect of the liquid-medium culture type on the synthesis of ligninolytic enzymes and the rate of decolorization of synthetic dyes by Irpex lacteus Culture type

Stationary culture Stationary culture with Tween 80 c Submerged culture Submerged culture with Tween 80 c

Residual dyea,b (%)

Maximal enzyme activity (Units lK1) MnP

Laccase

LiP

RO16

RBBR

76.2G4.4 386.0G8.5 0.2G0.1 53.0G2.4

2.0G0.0 0 1.0G0.1 0

1.1G0.2 0 0 0

14.2G0.7 12.4G1.0 84.0G1.8 13.1G1.8

0.3G0.4 ND 2.2G0.6 ND

Abbreviations: MnP, manganese-dependent peroxidase; LiP, lignin peroxidase; RO16, Reactive Orange 16 (azo dye); RBBR, Remazol Brilliant Blue R (anthraquinone dye); ND, not determined. a Initial dye concentrations, 150 mg lK1 (Z100%). b Decolorization measured after 7-d. c Tween 80 added to the medium at 1 g lK1.

residual dye concentration of 12–13% as was achieved in the stationary cultures (Table 1). The correlation between the decolorization of RO16 and a high level of extracellular MnP was confirmed in an experiment, where a 7-d-old agitated culture was subjected to stationary incubation (data not shown). The onset of efficient decolorization correlated with the rise of extracellular MnP activity that increased about 400-fold within 7 days. During this time, 95% of the initial dye amount of 150 mg lK1 was decolorized to demonstrate a correlation between the enzyme production and decolorization of the dye. No LAC and LiP activities were detected during the experiment. A correlation was observed between the ability of various white rot fungi to produce ligninolytic enzymes in LNMM stationary cultures and to remove PCBs from these cultures (Table 2). Irrespective of different combinations of ligninolytic enzymes present in culture fluids of the individual fungal organisms, there was an order of magnitude lower production of ligninolytic enzymes in P. ostreatus, compared to other fungi. This was correlated with the inability of P. ostreatus to remove PCBs from liquid media. A similar difference was observed when these fungi were used for decolorization of the anthraquinone dye RBBR and the polymeric dye Poly R-478 on agar plates (data not shown). The respective time periods necessary for a complete decolorization of the agar medium by P. ostreatus were 20 and 16 days. Similar periods determined for the other fungal organisms were only 6–10 and 5–10 days. The significant differences between the individual fungal organisms concerning the production of ligninolytic enzymes may exist under a range of growth conditions. This supposition has been used for selection of fungi with high biodegradative capabilities (Field et al., 1993; Novotny´ et al., 2001). 3.2. Immobilized fungal cultures A comparison of cultures of I. lacteus immobilized on an inert substrate (polyurethane) and a lignocellulosic substrate (pinewood) showed differences between the production of extracellular ligninolytic activities

(Table 3), comparable to those observed in stationary and submerged cultures (Table 1). The capacity for decolorization of RBBR was similar in both immobilized cultures which was consistent with the observation in liquid cultures (Table 1), where a significant reduction in the synthesis of MnP did not result in a decrease of RBBR decolorization rate. A similar decolorization efficiency of the two immobilized cultures was also observed in the case of textile coloring bath liquids containing dye mixtures Drimaren Blue and Drimaren Red (data not shown). The respective decolorization rates measured in the polyurethane foam reactor after 7-d were 83G6 and 94G4% of the initial absorbance value, compared to 99G1 and 82G9% in the pinewood reactor. In contrast, the ability to decolorise the coloring bath liquid containing the dye Acid Black correlated with a higher synthesis of MnP in the polyurethane foam bioreactor, where 95G3% of the initial dye absorbance was decolorized within 7-d, compared to only 18G9% in the pinewood reactor. Decolorization of the dye mixture Remazol Green was rather low in both bioreactors (26G5 and 45G4%, respectively) and not related to the MnP level produced. Table 2 Production of extracellular ligninolytic enzymes in stationary cultures of white rot fungi and their ability to remove PCBs in liquid medium Fungal organism

Controlb Phanerochaete chrysosporium Coriolopsis polyzona Trametes versicolor Pleurotus ostreatus a

Residual PCBsa

Maximal enzyme activity (Units lK1)a MnP

Laccase

LiP

mg per flask

%

– 101G12

– 0

– 77G18

3.2G0.5 2.4G1.4

100 75

56G9

22G8

13G6

1.9G0.6

59

28G8

14G3

0

1.6G0.7

50

0

3.2G0.3

100

2G0.3

1G0.1

Growth in liquid stationary cultures using LNMM medium for 3 weeks at 28 8C; initial concentration of the Delor 106 PCB mixture was 0.9 mg lK1. b One-week, heat-killed culture of P. chrysosporium.

ˇ . Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551 C

1549

Table 3 Decolorization of RBBR and extracellular enzyme activities in packed-bed bioreactors with Irpex lacteus immobilized on pinewood or polyurethane foam Time (days) 0 Polyurethane foam reactor Residual dye (%) 100.0G0.0 95.0G7.0 MnP (U lK1) 2.0G1.8 Laccase (U lK1) Pinewood cubes reactor Residual dye (%) 100.0G0.0 MnP (U lK1) 2.7G4.6 0.7G1.1 Laccase (U lK1)

2

4

6

10

71.2G8.2 83.3G56.9 1.3G1.2

45.1G17.9 64.3G13.1 1.3G2.3

14.2G3.8 52.3G54.6 2.0G3.5

2.9G2.6 77.7G15.7 3.3G5.8

37.1G11.2 12.0G2.0 2.0G2.0

4.3G5.1 18.3G3.8 1.3G2.3

0.0G0.0 12.3G9.3 2.3G3.2

0.0G0.0 10.0G8.7 0

Initial RBBR concentration was 150 mg lK1.

3.3. Soil fungal cultures Pre-sterilized soil was spiked with pyrene and anthracene and subsequently colonized by the explorative mycelium of the various fungi growing from the milled wheat straw substrate (Table 4). Ligninolytic enzymes were extracted from the soil using succinate/lactate buffer pH 4.5. The most extensive soil colonization was observed with P. ostreatus(11.0G1 mg ergosterol gK1 dry soil), the poorest with T. versicolor (2.2G0.4 mg ergosterol gK1 dry soil). Fungal mycelium secreted ligninolytic enzyme activities into soil (Table 4). Soil inoculated with P. ostreatus exhibited the highest levels of MnP and LAC and this correlated with the highest ability to remove anthracene and pyrene (Table 4). The ability to transform anthracene was confirmed by the accumulation of anthraquinone (data not shown). Negligible degradation rates were observed with T. versicolor in spite of significant ligninolytic enzyme levels secreted in the soil (Table 4). The colonization of soil by T. versicolor was, however, poor which may explain its inefficiency in PAH degradation (Pointing, 2001). P. chrysosporium exhibited the lowest enzyme levels, nonetheless was able to degrade pyrene. On the other hand, this fungus had a negligible impact on anthracene concentration (Table 4).

All synthetic dyes used were readily soluble in water. There was a strong correlation between the rate of decolorization of RO16 and Acid Black in the liquid medium cultures and the concentrations of ligninolytic enzymes (Table 1). A significant role of LiP, MnP and LAC in dye degradation by white rot fungi has been shown (McMullan et al., 2001) and the direct involvement of MnP and LAC in the decolorization of RO16 in I. lacteus cultures was demonstrated by its selective inhibition with sodium azide and n-propyl gallate (Svobodova´, unpublished). In our experiments, the efficient decolorizations of RBBR, Drimaren Blue and Drimaren Red were not directly dependent on high levels of ligninolytic enzymes (Tables 1 and 3). It is possible that other degradation systems could also be involved in decolorization, such as RBBR oxygenase and nonligninolytic systems (Pasti and Crawford, 1991; Vyas and Molitoris, 1995) or that low extracellular ligninolytic activities were sufficient for the rapid decolorization of the dyes in question.. The solubility of PCBs in water is generally very low, its value for 2,2 0 ,4,4 0 5,5 0 -hexachlorobiphenyl, a major compound whose concentration is highest of all hexachlorinated congeners characteristic of Delor 106, is 0.95 mg lK1 Table 4 Ligninolytic enzyme levels and the removal of anthracene and pyrene in spiked soil colonized by explorative mycelium of white rot fungi

4. Discussion Although a number of studies exist that deal with the involvement of peroxidases and laccases in biodegradation of recalcitrant compounds, not many of them focus on the relationship between the extracellular enzyme levels and degradation rates under in vivo conditions. This relationship can be obscured by the complexity of the biodegradation process, where various interactions and limitations may determine which step will become rate-limiting. Our study showed that in a majority of experiments using white rot fungi the degradation rates could be correlated with the levels of extracellular ligninolytic enzymes known to be involved. The work included a broad selection of recalcitrant compounds (dyes, PAHs, PCBs), fungal species and growth conditions.

Fungal organism

Maximal enzyme activity in soil (U gK1)a MnP

Laccase

Phanerochaete chrysosporium

0.03G0.00

0

Trametes versicolor Pleurotus ostreatus

0.18G0.13 0.20G0.13 0.65G0.17 0.25G0.06 Residual PAH concentration (mg kgK1) Anthraceneb Pyreneb 20.1G2.8 25.0G0.8 19.7G3.1 8.8G4.8 19.7G1.3 23.5G1.6 2.6G1.0 1.7G0.4

Aerated control Phanerochaere chrysosporium Trametes versicolor Pleurotus ostreatus

a The enzyme activity extracted from soil substrate is related to 1 g dry soil. b Initial concentration of each PAH was 50 mg kgK1 soil; PAH removal was measured after 8-week incubation at 24 8C under forced aeration.

1550

Cˇ. Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551

(Syracuse Research Corporation Phys Prop Database). Several species of white rot fungi were found to remove PCBs from culture media and in some of them, mineralization of 14C-labeled PCBs took place (Vyas et al., 1994; Yadav et al., 1995). The precise role of ligninolytic enzymes in this process is not clear (Pointing, 2001). High levels of ligninolytic enzymes in liquid cultures of T. versicolor and C. polyzona corresponded to the ability of these microorganisms to remove PCBs (Table 2). This relationship, however, did not apply to P. chrysosporium because of large differences in the residual PCB concentrations between the replicate treatments. Anthracene and pyrene are also strongly hydrophobic compounds but have been shown to be oxidized by MnP and LAC (Johannes et al., 1996; Gu¨nther et al., 1998). Other fungal enzyme systems such as membrane-associated cytochrome P-450 monooxygenase have also been found involved in PAH degradation (Sutherland et al., 1995; Bezalel et al., 1997). In the case of Bjerkandera adusta, the endogenous production of H2O2 and not the levels of MnP and LiP were reported to limit the rate of anthracene oxidation in a liquid medium (Kotterman et al., 1996). In our system, anthracene and pyrene were spiked into sterile soil, where sorption effects could also be expected (Providenti et al., 1993). The above-mentioned factors probably contributed to the poor correlation between the enzyme levels in soil and PAH degradation (Table 4). One can speculate about the importance of high levels of MnP and LAC in soil in P. ostreatus cultures as they showed the highest degradation rates (Table 4). We have no explanation for the poor performance in soil of T. versicolor, a strain that exhibited relatively high extracellular enzyme levels. However, this has already been reported by other authors (Martens and Zadrazˇil, 1998) and was reflected by the poor mycelial growth in our study.

5. Conclusions In spite of the existence of other factors known to limit the degradation of xenobiotics by white rot fungi in liquid media and soil (e.g. H2O2 production, limited bioavailability, hydrophobic properties of compounds), evidence was gathered that significant levels of extracellular ligninolytic enzymes was an important factor in ensuring high biodegradation rates. This was especially so in liquid media. MnP and to a lesser extent, LAC were shown to be the most important enzymes in our study. This can be different in fungal organisms possessing other combinations of extracellular ligninolytic activities (cf. fungal groups described by Hatakka, 1994). The importance of extensive growth and high levels of enzyme synthesis and secretion during remediation should be taken into account when bioremediation strategies are evaluated.

Acknowledgements We thank to Jitex Pisek a.s. for kindly providing us the samples of textile coloring bath liquids. The work was ˇ R 526/00/1303, KONTAKT supported by the projects GA C 2003-14 and 011-2004-05, and Institutional Research Concept AV0Z5020903.

References Bezalel, L., Hadar, Y., Cerniglia, C.E., 1997. Enzymatic mechanisms involved in phenanthrene degradation by the white rot fungus Pleurotus ostreatus. Applied and Environmental Microbiology 63, 2495–2501. Davis, M.W., Lamar, R.T., 1992. Evaluation of methods to extract ergosterol for quantitation of soil fungal biomass. Soil Biology & Biochemistry 24, 189–198. Fernando, T., Aust, S.D., 1994. Biodegradation of toxic chemicals by white rot fungi., in: Chaudhry, G.R. (Ed.), Biological Degradation and Bioremediation of Toxic Chemicals. Chapman & Hall, London, pp. 386–402. Field, J.A., De Jong, E., Feijoo-Costa, G., De Bont, J.A.M., 1993. Screening for ligninolytic fungi applicable to to the biodegradation of xenobiotics. Trends in Biotechnology 11, 44–48. Gramss, G., Kirsche, B., Voigt, K.-D., Gu¨nter, T., Fritsche, W., 1999. Conversion rates of five polycyclic aromatic hydrocarbons in liquid cultures of fifty-eight fungi and the concomitant production of oxidative enzymes. Mycological Research 103, 1009–1018. Gu¨nther, T., Sack, U., Hofrichter, M., Laetz, M., 1998. Oxidation of PAH and PAH-derivatives by fungal and plant oxidoreductases. Journal of Basic Microbiology 38, 113–122. Hammel, K.E., Green, B., Gai, W.Z., 1991. Ring fission of anthracene by a eukaryote. Proceedings of The National Academy of Sciences USA 88, 10605–10608. Hatakka, A., 1994. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiology Reviews 13, 125–135. Heinfling, A., Martı´nez, M.J., Martı´nez, A.T., Bergbauer, M., Szewzyk, U., 1998. Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganeseindependent reaction. Applied and Environmental Microbiology 64, 2788–2793. Johannes, C., Majcherczyk, A., Hu¨ttermann, A., 1996. Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds. Applied Microbiology and Biotechnology 46, 313–317. ˇ ., Svobodova´, K., Patel, K.C., Sˇasˇek, V., 2003. Kasinath, A., Novotny´, C Decolorization of synthetic dyes by Irpex lacteus in liquid cultures and packed-bed bioreactor. Enzyme Microbial Technology 32, 167–173. Ka¨stner, M., Streibich, S., Beyrer, M., Richnow, H.H., Fritsche, W., 1999. Formation of bound residues during microbial degradation of [!$C] anthracene in soil. Applied and Environmental Microbiology 65, 1834–1842. Kirk, T.K., Farrell, R.L., 1987. Enzymatic ‘combustion’: the microbial degradation of lignin. Annual Review of Microbiology 41, 465–505. Kotterman, M.J., Wasseveld, R.A., Field, J.A., 1996. Hydrogen peroxide production as a limiting factor in xenobiotic compound oxidation by nitrogen-sufficient cultures of Bjerkandera sp. strain BOS55 overproducing peroxidases. Applied and Environmental Microbiology 62, 880–885. Leisola, M.S.A., Fiechter, A., 1985. Ligninase production in agitated conditions by Phanerochaete chrysosporium. FEMS Microbiology Letters 29, 33–36.

ˇ . Novotny´ et al. / Soil Biology & Biochemistry 36 (2004) 1545–1551 C Lin, J.E., Wang, H.Y., Hickey, R.F., 1990. Degradation kinetics of pentachlorophenol by Phanerochaete chrysosporium. Biotechnology and Bioengineering 35, 1125–1134. Martens, R., Zadrazˇil, F., 1998. Screening of white rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil. Folia Microbiologica 43, 97–103. McMullan, G., Meehan, C., Conneely, A., Kirby, N., Robinson, T., Nigam, P., Banat, I.M., Marchant, R., Smyth, W.F., 2001. Microbial decolourisation and degradation of textile dyes. Applied Microbiology and Biotechnology 56, 81–87. Niku-Paavola, M.L., Karhunen, P., Salola, P., Raunio, V., 1988. Ligninolytic enzymes of the white rot fungus Phlebia radiata. Biochemical Journal 254, 877–884. ˇ ., Vyas, B.R.M., Erbanova´, P., Kuba´tova´, A., Sˇasˇek, V., 1997. Novotny´, C Removal of PCBs by various white rot fungi in liquid cultures. Folia Microbiologica 42, 136–140. ˇ ., Erbanova´, P., Sˇasˇek, V., Kuba´tova´, A., Cajthaml, T., Lang, E., Novotny´, C Krahl, J., Zadrazˇil, F., 1999. Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi. Biodegradation 10, 159–168. ˇ ., Erbanova´, P., Cajthaml, T., Rothschild, N., Dosoretz, C., Novotny´, C Sˇasˇek, V., 2000. Irpex lacteus, a white rot fungus applicable to water and soil bioremediation. Applied Microbiology and Biotechnology 54, 850–853. ˇ ., Rawal, B., Bhatt, M., Patel, M., Sˇasˇek, V., Molitoris, H.P., Novotny´, C 2001. Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes. Journal of Biotechnology 89, 113– 122. Ollikka, P., Alhonma¨ki, K., Leppa¨nen, V.M., Glumoff, T., Raijola, T., Suominen, I., 1993. Decolorization of azo, triphenylmethane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Applied and Environmental Microbiology 59, 4010–4016. Orth, A.B., Tien, M., 1995. Biotechnology of lignin degradation, in: Esser, K., Lemke, P.A. (Eds.), The Mycota. II. Genetics and Biotechnology . Springer, Berlin, pp. 287–302. Pasti, M.B., Crawford, D.L., 1991. Relationship between the abilities of streptomycetes to decolorize three anthrone-type dyes and to degrade hemicellulose. Canadian Journal of Microbiology 37, 902–907. Pointing, S.B., 2001. Feasibility of bioremediation by white-rot fungi. Applied Microbiology and Biotechnology 57, 20–33.

1551

Providenti, M.A., Lee, H., Trevors, J.T., 1993. Selected factors limiting the microbial degradation of recalcitrant compounds. Journal of Industrial Microbiology 12, 379–395. Ru¨ttmann-Johnson, C., Lamar, R.T., 1997. Binding of pentachlorophenol to humic substances in soil by the action of white rot fungi. Soil Biology & Biochemistry 29, 1143–1148. Stotzky, G., Burns, R.G., 1982. The soil environment: clay–humus– microbe interactions, in: Burns, R.G., Slater, J.H. (Eds.), Experimental Microbial Ecology . Blackwell Scientific Publications, Oxford, London, pp. 105–133. Sutherland, J.B., Rafii, F., Khan, A., Cerniglia, C.E., 1995. Mechanisms of polycyclic aromatic hydrocarbon degradation, in: Young, L.Y., Cerniglia, C.E. (Eds.), Microbial Transformations and Degradation of Toxic Organic Chemicals. Wiley, New York, pp. 269–306. Syracuse Research Corporation Phys Prop Database, http://esc.syrres.com. Tanaka, H., Itakura, S., Enoki, A., 1999. Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor. Journal of Biotechnology 74, 57–70. Thurston, C.F., 1994. The structure and function of fungal laccases. Microbiology 140, 19–26. Tien, M., Kirk, T.K., 1988. Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymology 161, 238–249. VanAcken, B., Godefroid, L.M., Peres, C.M., Naveau, H., Agathos, S.N., 1999. Mineralization of 14C-U ring labeled 4-hydroxylamino-2,6dinitrotoluene by manganese-dependent peroxidase of the white-rot basidiomycete Phlebia radiata. Journal of Biotechnology 68, 159–169. Vyas, B.R.M., Molitoris, H.P., 1995. Involvement of an extracellular H2O2dependent ligninolytic activity of the white rot fungus Pleurotus ostreatus in the decolorization of Remazol Brilliant Blue R. Applied and Environmental Microbiology 61, 3919–3927. Vyas, B.R.M., Bakowski, S., Sˇasˇek, V., Matucha, M., 1994. Degradation of anthracene by selected white rot fungi. FEMS Microbiology Letters 14, 65–70. Wang, Y., Yu, J., 1998. Adsorption and degradation of synthetic dyes on the mycelium of Trametes versicolor. Water Science and Technology 38, 233–238. Yadav, J.S., Quensen III., J.F., Tiedje, J.M., Reddy, C.A., 1995. Degradation of polychlorinated biphenyl mixtures (Aroclors 1242, 1254 and 1260) by the white rot fungus Phanerochaete chrysosporium as evidenced by congener specific analysis. Applied and Environmental Microbiology 61, 2560–2565.