1 Department of Groundwater Microbiology, UFZ Centre for Environmental Research Leipzig-Halle, Theodor-Lieser-Strasse 4, D-06120 Halle, Germany
2 Department of Analytical Chemistry, UFZ Centre for Environmental Research Leipzig-Halle, D-04318 Leipzig, Germany
Correspondence
Dietmar Schlosser
dietmar.schlosser{at}ufz.de
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ABSTRACT |
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INTRODUCTION |
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Environmentally ubiquitous mitosporic fungi occur in surface water (de Lope & Sanchez, 2002; Niewolak, 1975
; Nikolcheva & Bärlocher, 2004
; Tóthová, 1999
). Terrestrial isolates of such organisms are known to degrade e.g. polycyclic aromatic hydrocarbons (Cerniglia & Sutherland, 2001
), fungicides (Gesell et al., 2001
) and chlorophenols (Hofrichter et al., 1994
), suggesting that mitosporic fungi may also contribute to the microbial degradation of other organic contaminants present in aquatic environments. Aquatic hyphomycetes are a phylogenetically diverse group of mitosporic fungi specifically adapted to aquatic environments. They initiate the decomposition of organic matter in rivers, streams and lakes by colonilization of plant detritus arising from the riparian vegetation (Bärlocher, 1992
; Nikolcheva et al., 2003
). Although these fungi are most common in unpolluted, oxygen-rich water, and are sensitive to pollution (Bärlocher, 1992
), several strains of aquatic hyphomycetes have also been isolated from contaminated surface and groundwater in recent years (Krauss et al., 2001
, 2003a
, b
; Sridhar & Raviraja, 2001
). However, the knowledge about the potential of these organisms to degrade organic xenobiotics is limited to a few examples (Hodkinson, 1976
).
Laccases (EC 1.10.3.2) are extracellular multicopper-containing oxidoreductases most prominent in white-rot basidiomycetes, where they are believed to contribute to the biodegradation of lignin. These enzymes are also well known from ascomycetes, imperfect fungi and yeasts (Thurston, 1994). Laccases unspecifically couple the one-electron oxidation of reducing substrates to the reduction of dioxygen to water and oxidize e.g. different phenolic compounds and aromatic amines (Xu, 1996
). Their substrate range can be considerably extended in the presence of small, diffusible compounds acting as redox mediators (Bourbonnais & Paice, 1990
), particularly in white-rot fungi (Eggert et al., 1996
; Johannes & Majcherczyk, 2000
). The oxidation of nonylphenol and bisphenol A, another phenolic environmental pollutant with endocrine activity, by laccases from white-rot basidiomycetes (Fukuda et al., 2001
; Tsutsumi et al., 2001
; Uchida et al., 2001
) and soil-derived ascomycetes (Saito et al., 2003
) has previously been demonstrated. Both pollutants are also degraded by the extracellular lignin-modifying enzyme manganese peroxidase (EC 1.11.1.13) produced by white-rot fungi (Tsutsumi et al., 2001
).
Here, we focus on the degradation of t-NP by a strain of the aquatic hyphomycete Clavariopsis aquatica, a species frequently observed in rivers and streams (Baldy et al., 2002; Krauss et al., 2001
, 2003a
; Nikolcheva et al., 2003
), and a strain of a mitosporic fungus isolated from river water containing t-NP as a contaminant. In addition, 4-n-NP was employed in some experiments as a structurally defined reference compound. To our knowledge, the degradation of nonylphenol by fungi isolated from aquatic environments has not previously been described. We also address the potential role of laccases, produced by both of the fungal strains, in the biodegradation of nonylphenol.
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METHODS |
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Isolation, identification and maintenance of fungal strains.
Strain UHH 1-6-18-4 was isolated in April 2002 from water of the Saale river close to the village of Lettin, which is located at the northern city district boundary of Halle/Saale (central Germany). At the sampling site, t-NP concentrations in river water of about 260, 154 and 206 ng l1 were determined during sampling campaigns in February 2002, March 2003 and September 2003, respectively. Water samples (150 µl) were poured onto agar plates containing 1 % (w/v) malt extract, 1·5 % (w/v) agar, 0·9 % NaCl, 400 000 units penicillin l1 and 0·4 g streptomycin l1 (pH 5·65·8) (Krauss et al., 2001), which were incubated at 14 °C for several days. Pure cultures were obtained by transferring single colonies onto fresh agar plates. To avoid undesired sporulation, isolate UHH 1-6-18-4 was routinely maintained in liquid culture, using Erlenmeyer flasks (250 ml) containing 75 ml of a 1 % (w/v) malt extract medium (pH 5·65·8). Liquid cultures were incubated at 14 °C and 120 r.p.m. in the dark, and new subcultures were established every 4 weeks. Strain UHH 1-6-18-4 was submitted to the German Collection for Micro-organisms and Cell Cultures (DSMZ, Braunschweig, Germany) for identification. The isolate forms discrete hyaline conidiophores with penicillioid branching, flask-like phialides with a prominent, funnel-shaped collarette and nearly globose apiculate conidia in slimy heads. A brownish orange pigment diffuses into the agar medium. These characteristics point to an affinity with the polyphyletic genus Phialophora as described by Gams (2000)
, but the isolate does not fit any of the species listed there (P. Hoffmann, personal communication).
Clavariopsis aquatica de Wild. strain WD(A)-00-1 was isolated from the brook Steinbach near Waldau (district of Zeitz, Saxony-Anhalt, central Germany), which continuously receives leachates containing tar oil residues arising from the historical disposal of wastes of the former lignite-processing industries in the region. Fallen leaves of alder (Alnus glutinosa) were collected in the botanical garden of the Martin-Luther-University Halle-Wittenberg and air-dried, and autoclaved leaf discs (diameter 1·5 cm) were placed in nylon mesh bags (10x10 cm, 1 mm mesh) and exposed in the brook for 4 weeks in June 2001. Leaf discs recovered from the stream were placed in sterile Petri dishes containing distilled water (48 leaf discs per Petri dish), and incubated at 10 °C. After 26 days, Petri dishes were screened for conidiophores under a stereomicroscope. To isolate pure cultures, individual spores were picked up with capillary pipettes, and aseptically transferred to the penicillin- and streptomycin-containing malt agar plates described above. C. aquatica was identified based on its characteristic spore shape by L. Maranová (Czech Collection of Microorganisms, Masaryk University, Brno, Czech Republic). The fungus was maintained at 14 °C on malt agar plates containing 1 % (w/v) malt extract and 1·5 % (w/v) agar.
Nonylphenol-degradation experiments.
Liquid culture experiments were conducted in Erlenmeyer flasks (125 ml) containing 37·5 ml of the malt extract medium described above. Flasks were inoculated with either 0·5 ml of a mycelial suspension, prepared by homogenizing 10 agar plugs (7 mm diameter) derived from the margins of well-grown agar plate cultures of C. aquatica together with 10 ml sterile distilled water, or 0·5 ml of a mycelial suspension derived from UHH 1-6-18-4 liquid cultures at the onset of the stationary growth phase. Fungal cultures were incubated with agitation (120 r.p.m.) at 14 °C in the dark. t-NP and 4-n-NP were aseptically added from 25 mM stock solutions in methanol to give final concentrations of 250 µM [corresponding to 1 % (v/v) methanol] and 100 µM (corresponding to 0·4 % methanol), respectively, at the time points indicated in the text.
Laccase-containing concentrated crude culture supernatants of both fungi (see below) served as enzyme sources in experiments on the enzymic degradation of t-NP and 4-n-NP. Reaction mixtures consisted of 0·2 U laccase ml1, t-NP or 4-n-NP at concentrations described in the text (added from 25 mM stock solutions) and 0·1 % (w/v) Tween 80 to improve the solubility of nonylphenols, in 100 mM sodium citrate buffer (pH 4·0). Controls contained laccase preparations that were heat-inactivated by autoclaving at 120 °C for 20 min. Enzyme incubations were carried out with agitation (120 r.p.m.) at 24 °C in the dark.
Analysis of nonylphenols and degradation products.
Nonylphenol concentrations in fungal culture supernatants and enzymic degradation experiments were determined by HPLC directly without separation of organic and aqueous phases. Samples (1 ml, cell-free in the case of fungal cultures) were taken at the time points indicated in the text. After addition of an equal volume of methanol and vigorous mixing, samples were centrifuged for 30 min at 20 817 g and 4 °C. Supernatants were analysed on a Merck-Hitachi HPLC system consisting of an L-7120 pump, an L-7200 autosampler, an L-7420 UV/VIS detector and a LiChrospher 100 RP 18-5 column (Merck-Hitachi). Gradient elution started with 20 % (v/v) acetonitrile in distilled water, which was kept constant for 2 min, followed by a linear increase to 90 % acetonitrile within 10 min. The 90 % acetonitrile concentration was kept constant for 3 min, then linearly decreased back to 20 % within 5 min, and kept constant for another 5 min. The flow rate was 1 ml min1, and the detection wavelength was 277 nm. The retention time for 4-n-NP was 14·8 min under these conditions. The t-NP mixture eluted as a single peak at 13·9 min. The method was calibrated with external standards.
To resolve t-NP isomers and to detect nonylphenol degradation products by GC-MS, fungal cultures were harvested at the time points indicated in the text and extracted with 62·5 ml ethyl acetate following the simultaneous disintegration of fungal mycelia for 1 min, using a mechanical homogenizer (Ultraturrax; IKA) at 24 000 r.p.m. Ethyl acetate fractions were dried over anhydrous sodium sulfate and evaporated to dryness. Residues were redissolved in 8 ml methanol and subjected to either HPLC or GC-MS analysis. In addition, derivatization of possible H-acidic compounds was carried out with bis(trimethylsilyl)trifluoroacetamide (BSTFA; Merck) after evaporation of the ethyl acetate extracts to dryness (Nakagawa et al., 2001). GC-MS analysis was performed on a 6890 gas chromatograph equipped with a 5973 mass-sensitive detector (Agilent Technologies) and an HP-5 MS fused-silica column (30 mx0·25 mm i.d., 0·25 µm film thickness; Agilent Technologies) as previously described (Braun et al., 2003
). Analysis was conducted in full-scan mode over an m/z range of 50 to 500 amu. Spectral interpretation was aided by the National Institute of Standards and Technology (NIST) 98 mass spectral library stored in the GC-MS controller unit.
The t-NP concentration in river water was determined by GC-MS after solid-phase microextraction, as previously described (Braun et al., 2003).
Gel-permeation chromatography (GPC) was employed in experiments with concentrated laccases, to detect nonylphenol degradation products with higher molecular masses than that of the parent compound. Samples (1·5 ml) were acidified with 150 µl 1 M HCl and centrifuged for 30 min at 20 817 g and 4 °C. Supernatants were discarded; pellets were redissolved in 500 µl 0·1 M NaOH and subjected to GPC. For GPC analysis the same HPLC system was used as described above, except that an L-4755 diode-array detector (Merck-Hitachi) was employed and separations were carried out on a HEMA Bio-40 GPC column (8x300 mm; PSS GmbH). The solvent system consisted of 20 % (v/v) acetonitrile, 80 % (v/v) distilled water, NaNO3 (5 g l1), and K2HPO4 (2 g l1). Isocratic elution started at a flow rate of 1 ml min1, which was kept constant for 3 min. After this, the flow rate was linearly decreased to 0·3 ml min1 within 3 min, and kept at this level for another 20 min. Then it was linearly raised back to 1 ml min1 within 2 min and held for another 5 min. The absorbance was monitored over a wavelength range of 225600 nm. For estimation of molecular masses, retention times of degradation products were compared with that of polystyrene sulfonate sodium salt molecular mass standards (PSS GmbH).
Production and concentration of laccases.
C. aquatica was grown in a laboratory fermenter (Biostat MD; Braun Biotech International) in discontinuous culture. The cultivation medium (2 l volume) consisted of the liquid malt extract medium and was inoculated with 100 ml of a homogenized liquid culture of C. aquatica at the early phase of stationary growth. The fermenter was operated at 14 °C, an aeration rate of 1 l sterilized air min1, and a stirring speed of 120 r.p.m. A temperature of 14 °C was chosen because it was more favourable for laccase production than higher temperatures (data not shown). UHH 1-6-18-4 was grown in Erlenmeyer flasks as already described. For both fungi, laccase production was stimulated by addition of a mixture of 1 mM vanillic acid and 50 µM CuSO4 during the exponential growth phase. After reaching maximum laccase activities as monitored by regular enzyme activity measurements, fungal cultures were harvested, and the fungal mycelia were removed by filtration through cellulose nitrate filters (0·45 µm pore size; Sartorius). Cell-free culture supernatants were concentrated with a tangential-flow membrane filter (polysulfone minisette, 10 kDa cut-off; Pall Filtron), and washed several times with 10 mM sodium acetate buffer (pH 5·5).
Enzymic determinations.
Malt agar plates containing 2 mM ABTS in addition were used to indicate the production of laccase or peroxidases. Plates were inoculated by placing one agar plug derived from the edge of well-grown fungal cultures on agar plates into the centre of a plate. The development of fungal mycelia and the concomitant colorization of the ABTS plates was followed over several days.
Laccase activities in liquid cultures were determined by following the oxidation of 3 mM ABTS in 100 mM sodium citrate buffer (pH 4·0) at 420 nm (420=36 mM 1 cm1; Johannes & Majcherczyk, 2000
), using a microplate reader (SLT Spectra; Tecan). Enzyme activities are expressed as units, where 1 U=1 µmol product formed min1.
Gel electrophoresis and staining.
SDS-PAGE was performed on 10 % polyacrylamide gels (Laemmli, 1970), using a Mini PROTEAN 3 electrophoresis chamber (Bio-Rad). To provide conditions that allow laccase activity staining (subsequently referred to as SDS-PAGE under non-denaturing and non-reducing conditions),
-mercaptoethanol was omitted from the sample buffer, and samples were not boiled prior to loading onto the gel. After the run was complete, the gel was cut to separate lanes containing laccase from those containing molecular mass markers. Laccase-containing lanes were stained for enzyme activity with 3 mM ABTS (Höfer & Schlosser, 1999
), and lanes containing molecular mass markers were stained with Coomassie brilliant blue R-250 (Bio-Rad) for protein. A semi-logarithmic plot of molecular masses of standard proteins versus their relative migration distances revealed a linear dependency with a correlation coefficient >0·99, thus allowing the alignment of apparent molecular masses of laccase activity bands. In addition, proteins on gels containing laccase-active samples from concentrated culture supernatants and molecular mass markers were visualized by silver staining.
Determination of fungal dry weights.
Fungal cultures were harvested at the time points indicated in the text; mycelia were removed from fungal cultures by filtration through filter papers (Whatman no. 6), washed with 50 ml distilled water, dried at 80 °C for 24 h, and weighed.
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RESULTS |
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Degradation of 4-n-NP was followed by addition of 100 µM of the compound to liquid cultures of UHH 1-6-18-4 and C. aquatica pregrown for 5 days. Tween 80 was omitted. Control cultures were inactivated by adding sodium azide (500 mg l1) at culture day 5. Additional controls consisted of uninoculated culture medium and 4-n-NP. Living fungal cultures and respective controls were incubated for another 42 days, harvested, extracted with ethyl acetate, and extracts were subjected to HPLC. 4-n-NP was completely removed by active UHH 1-6-18-4 cultures and had essentially completely disappeared in C. aquatica cultures, where a remaining concentration of 1±0 µM (mean±standard deviation from triplicate cultures) was detected. The 4-n-NP concentration in uninoculated controls was 113±2 µM (mean±standard deviation from triplicate experiments), indicating complete recovery. Azide-inactivated cultures yielded incomplete 4-n-NP recoveries of 49±5 and 79±3 µM (means±standard deviations from triplicate cultures) for UHH 1-6-18-4 and C. aquatica, respectively, possibly due to remaining degradation activities or strong binding to the fungal biomass. GC-MS analysis of ethyl acetate extracts of active cultures could not detect previously described 4-n-NP degradation metabolites such as 9-(4-hydroxyphenyl)nonanoic acid, 3-(4-hydroxyphenyl)propionic acid, 4-hydroxycinnamic acid, 4-hydroxyacetophenone, 4-aminoacetophenone or 4-hydroxybenzoic acid (Thibaut et al., 1999; Vallini et al., 2001
; Yuan et al., 2004
). No peaks potentially representing other degradation products were detected, which may indicate complete degradation of 4-n-NP by both of the fungal strains.
Production of laccases by strain UHH 1-6-18-4 and C. aquatica
On ABTS-containing malt agar plates, blue-green zones around the developing fungal mycelia indicated the formation of the ABTS cation radical. The reaction was clearly detectable at essentially comparable intensities with both fungi and covered a radius of approximately 2·5 cm after 5 days, in each case.
Representative time-courses of laccase activities in culture media of liquid UHH 1-6-18-4 and C. aquatica cultures, as well as corresponding fungal dry weights, are shown in Fig. 5(a) and (b), respectively. In a total of four additional experiments conducted under identical conditions, laccase peak titres appeared between 7 and 24 days (UHH 1-6-18-4) and 12 and 17 days (C. aquatica) of cultivation, and varied between approximately 11 and 30 U l1 in UHH 1-6-18-4 and about 0·3 and 1 U l1 in C. aquatica (data not shown). Thus, C. aquatica secretes very little laccase into the culture medium in the absence of inducers whereas UHH 1-6-18-4 produces substantial amounts of extracellular laccase under such conditions. Both t-NP and 4-n-NP were previously shown to enhance laccase activities in the white-rot fungus Trametes versicolor (Kollmann et al., 2003
). Because of its special environmental relevance, we assessed the effect of t-NP on laccase production and fungal growth of UHH 1-6-18-4 and C. aquatica under the degradative culture conditions employed. In both fungi, addition of 250 µM t-NP had no effect on laccase activities or fungal growth (data not shown). In contrast, the addition of a mixture of 50 µM Cu2+ (in the form of CuSO4) and 1 mM vanillic acid clearly enhanced laccase titres in UHH 1-6-18-4 up to approximately 125 U l1 (Fig. 5a
), and in C. aquatica up to about 250 U l1 (Fig. 5b
). The fungal dry weights were not affected by the addition of these compounds (data not shown). Manganese peroxidase, manganese-independent peroxidase (peroxidase, EC 1.11.1.7) and lignin peroxidase (EC 1.11.1.14) activities were assessed as previously described (Höfer & Schlosser, 1999
; Schlosser et al., 1997
) but were not observed during any of the experiments.
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DISCUSSION |
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Our results show that a mitosporic fungal strain isolated from nonylphenol-contaminated river water, and a strain of the aquatic hyphomycete C. aquatica, degraded all of the t-NP constituents analytically resolved under our experimental conditions, to individual extents (Fig. 2). The biodegradative capabilities of C. aquatica have already been demonstrated in a previous study, with the fungus being reported to degrade the insecticide 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) to some extent (Dalton & Smith, 1971
). Degradation of t-NP was accompanied by the simultaneous formation of several metabolites, and GC-MS analysis of these products implies that either subterminal or terminal hydroxylation had occurred at the respective nonyl moiety of individual t-NP isomers (Fig. 3
, Table 1
), due to the action of as yet unknown intracellular hydroxylating enzymes. We are not aware of studies describing aromatic metabolites that arise from degradation of complex mixtures of different t-NP isomers with branched side chains by micro-organisms. The detection of phenolic t-NP degradation products with shortened alkyl chains (Fig. 4
, Table 1
) and the metabolite 4-hydroxybenzoic acid identified in UHH 1-6-18-4 (Table 1
) imply subsequent breakdown of hydroxylated, branched nonyl chains of certain t-NP isomers. This may be restricted to t-NP constituents containing less branched nonyl chains, as suggested by the base peak ion at m/z 135 observed in mass spectra of the t-NP degradation metabolites with shortened alkyl chains (molecular ion at m/z 208) and their lower numbers as compared to those of hydroxynonyl phenol metabolites (Table 1
).
UHH 1-6-18-4 produced extracellular laccase under degradative culture conditions, whereas extracellular laccase titres in the absence of appropriate agents were negligible in C. aquatica (Fig. 5). A mixture of Cu2+, which is known to induce differential laccase gene expression in asco- and basidiomycetes (Litvintseva & Henson, 2002
; Soden & Dobson, 2001
), and the lignin-related compound vanillic acid enhanced laccase production in both fungi investigated (Fig. 5
). The concomitant occurrence of different laccase forms as observed (Fig. 6
) is a well-known phenomenon in filamentous fungi and may be attributed to multiple laccase genes or post-translational modification (Litvintseva & Henson, 2002
; Palmieri et al., 2003
; Thurston, 1994
). C. aquatica has been connected to ascomycetous teleomorphs (Nikolcheva & Bärlocher, 2004
; Webster, 1992
). In ascomycetes, laccases have frequently been described (Kiiskinen et al., 2002
; Litvintseva & Henson, 2002
; Saito et al., 2003
). Fungi growing on plant material in aquatic environments would be expected to produce laccase under natural conditions; this could also apply to C. aquatica, which did not excrete substantial amounts of laccase under the nonylphenol-degradation conditions employed within the present study (Fig. 5
).
Cell-free laccase-containing crude culture liquids of both fungi degraded t-NP as well as 4-n-NP (Table 2). The efficiency of t-NP degradation was considerably enhanced in the presence of the model redox mediator ABTS. For nonylphenol and bisphenol A oxidation catalysed by laccase from a soil-derived ascomycete, Km values of 5 and 10 mM, respectively, were reported (Saito et al., 2003
). Such high Km values would be rather unfavourable for an efficient removal of these contaminants by laccases in aquatic ecosystems, considering the low pollutant concentrations observed in such environments (Heemken et al., 2001
; Kolpin et al., 2002
; Stachel et al., 2003
; Ying et al., 2002
). However, the reported accumulation of nonylphenol and bisphenol A on water and sediment particles (Heemken et al., 2001
; Ying et al., 2002
), as well as on organic surfaces (Takahashi et al., 2003
), may help to overcome kinetic limitations. For nonylphenol, bioaccumulation factors of up to 650 and 990 were reported for river periphytons and benthos, respectively, where bisphenol A was found to be concentrated up to 650- and 170-fold (Takahashi et al., 2003
). Also, laccases from aquatic fungi may have kinetic features differing from those of terrestrial species; this needs to be elucidated.
Laccase-catalysed degradation of t-NP led to the formation of products with higher molecular masses than that of the parent compound (Fig. 7), indicating oxidative coupling of primary oxidation metabolites. Similarly, the formation of polymerization products was reported for nonylphenol and bisphenol A degradation by laccases and manganese peroxidases from white-rot fungi (Tsutsumi et al., 2001
; Uchida et al., 2001
), which led to the removal of the oestrogenic activities of the contaminants (Tsutsumi et al., 2001
). In aquatic ecosystems, laccase-catalysed degradation of endocrine disruptors may lead to their removal from these environments by formation of bound residues with organic matter, as already proposed for oxidoreductase-catalysed degradation of chlorophenols in presence of stream fulvic acids (Sarkar et al., 1988
).
Together with the results from experiments with whole fungal cultures, the enzymic degradation experiments suggest that an additional extracellular, laccase-based degradation mechanism had contributed to t-NP degradation by UHH 1-6-18-4 under the laboratory conditions employed within the present study. This is supported by the observation that t-NP was more efficiently degraded by UHH 1-6-18-4 than by C. aquatica (Figs 1 and 2). A comparison of the fungal dry weights of both strains rules out the possibility that this effect was simply due to a possibly higher biomass of UHH 1-6-18-4. Instead, the opposite was observed (Fig. 5
). Also, since laccases are known to unspecifically degrade a broad variety of phenolic compounds, a laccase-catalysed t-NP degradation could be responsible for the lower specificity of degradation of individual t-NP isomers observed in UHH 1-6-18-4 (Fig. 2
). Laccase additionally may be involved in further degradation of secreted phenolic t-NP degradation metabolites. This is supported by the peak intensities of these compounds, which were considerably lower in UHH 1-6-18-4 than in C. aquatica (Figs 3, 4
). Similarly, laccase may contribute to t-NP degradation by C. aquatica in its natural aquatic environment.
In conclusion, the results of the present study support a possible role for fungi living in aquatic ecosystems in degradation of water contaminants with endocrine activity. Furthermore, they emphasize two different mechanisms simultaneously employed by aquatic fungi to initiate t-NP degradation: intracellular hydroxylation of individual t-NP isomers at their branched nonyl chains and subsequent side-chain degradation of certain isomers; and extracellular attack of t-NP by laccase, the latter leading to oxidative coupling of primary radical products to compounds with higher molecular masses. Besides the potential role of such degradation processes for natural attenuation processes in freshwater environments, this also offers new perspectives for biotechnological applications such as wastewater treatment.
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ACKNOWLEDGEMENTS |
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Received 24 June 2004;
revised 12 October 2004;
accepted 13 October 2004.
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