quantiﬁcation of the inﬂuence of extracellular laccase and ... · (np112) were about 1.6- and...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2009, p. 4398–4409 Vol. 75, No. 130099-2240/09/$08.00�0 doi:10.1128/AEM.00139-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Quantification of the Influence of Extracellular Laccase and IntracellularReactions on the Isomer-Specific Biotransformation of the

Xenoestrogen Technical Nonylphenol by the AquaticHyphomycete Clavariopsis aquatica�

Claudia Martin,1 Philippe F. X. Corvini,2 Ralph Vinken,3 Charles Junghanns,1Gudrun Krauss,1 and Dietmar Schlosser1*

UFZ, Department of Environmental Microbiology, Helmholtz Centre for Environmental Research-UFZ, 04318 Leipzig, Germany1;Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, 4132 Muttenz,

Switzerland2; and Dr. Knoell Consult GmbH, 51377 Leverkusen, Germany3

Received 20 January 2009/Accepted 29 April 2009

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase andintracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol(NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selectivesuppression of either laccase or intracellular reactions resulted in essentially comparable maximal removalrates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained fromlaccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reactionmixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomersby bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast withlaccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric compositionof remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficientlydegraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern ofthe quaternary �-carbon of t-NP isomers does not seem to be very important for intracellular transformationin C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectraldata of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimeth-ylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, andthe subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturationand methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.

Nonylphenol ethoxylates (NPEOs) represent a major group ofindustrial nonionic surfactants. Technical nonylphenol (t-NP),used for the production of NPEOs, is synthesized by Friedel-Crafts alkylation of phenol with a mixture of differently branchednonenes. It therefore comprises a great variety of mainly para-substituted isomers, with variously branched nonyl chains. About50 to 80 t-NP isomers were estimated to occur in environmentallyrelevant matrices (19). The incomplete bioconversion of NPEOsin wastewater treatment plants results in the formation of the lessbiodegradable t-NP and is considered a major source of thiscontaminant in the aquatic environment (57). The recalcitranceof t-NP to biodegradation is partly due to the presence in morethan 85% of the t-NP isomers of a quaternary �-carbon in thebranched nonyl chain. Such structural characteristics are consid-ered to limit biological nonyl chain oxidation (11, 53, 55). Non-ylphenols are known to disrupt normal endocrine functions invertebrates (57). Certain isomers contained in t-NP have beenreported to possess a considerably higher estrogenic activity thanthe t-NP mixture (15). Due to increasing concerns with respect to

their largely unknown environmental fate and potentially adverseenvironmental and human health effects, nonylphenols have beenlisted as priority hazardous substances in the EU water frame-work directive.

In light of the concerns above, microbial reactions with thepotential to reduce nonylphenol concentrations in the environ-ment but also offering new possibilities for applications such aseffluent treatment have received increasing attention (11).Among environmental microorganisms, both aquatic and ter-restrial fungi, as well as bacteria, have been shown to degradet-NP (11). Fungal attack on nonylphenols differs from bacterialnonylphenol degradation. In the case of intracellular nonyl-phenol biotransformation reactions catalyzed by fungi, onlymetabolites modified in the alkyl chain have been described(23, 52). Metabolites indicative of oxidation of the phenolicring have not been described to date. Bacterial degradationpathways have only been documented in the genera Sphin-gomonas and Sphingobium. Bacterial mineralization of the ar-omatic moiety of t-NP isomers to CO2 and H2O is initiated viaring hydroxylation at the ipso (C-4) position of the phenolicring, and nonanols are produced from the nonyl chains (10, 11,15, 16). Bacteria have been shown to utilize branched-chainnonylphenols as growth substrates (11, 12, 17, 43). In contrast,only one report describes the growth of a fungus, the yeast

* Corresponding author. Mailing address: Department of Environ-mental Microbiology, Helmholtz Centre for Environmental Research-UFZ, Permoserstrasse 15, 04318 Leipzig, Germany. Phone: 49 341 2351329. Fax: 49 341 235 2247. E-mail: [email protected].

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Candida aquaetextoris, on nonylphenol (the isomer 4-n-NPcontaining a linear nonyl chain) (52). With respect to fungalattack on t-NP, cometabolism seems to be the dominatingprocess (11). Recent literature data indicate that certain t-NPisomers with an estrogenic potency higher than those of theoriginal t-NP mixture can be enriched in remaining t-NP. Thisresults from the selective removal of individual isomers uponbacterial ipso-substitution degradation mechanisms (15). How-ever, the effects of fungal biotransformation reactions on theisomeric profile of t-NP have not yet been quantified.

Laccases are extracellular multicopper oxidases. These havemost frequently been described in white-rot basidiomycetes,which unspecifically oxidize via one-electron abstraction cer-tain lignin constituents, as well many xenobiotic compounds.Thereby, organic radicals are generated as the primary oxida-tion products (3). Among the several groups of fungi found inaquatic environments, aquatic hyphomycetes (AQH) are aphylogenetically diverse group of mitosporic fungi specificallyadapted to their exclusively aquatic lifestyle. AQH have beenshown to metabolize several organic environmental pollutants,including t-NP (23), polycyclic musk fragrances (31), pesticidemetabolites (2), and synthetic dyes (22). Therefore, with re-spect to the fungal attack on organic pollutants found inaquatic ecosystems, AQH are of special importance. Laccaseproduction by strictly aquatic fungi such as AQH has alreadybeen demonstrated and discussed in the context of lignocellu-lose decay in aquatic ecosystems (1). A role of this enzyme inthe AQH-catalyzed breakdown of aquatic environmental pol-lutants has been recently suggested. Here, laccase isolatedfrom the AQH Clavariopsis aquatica was shown to act onnonylphenol (23) and polycyclic musk fragrances (31). Laccasehas also been implicated in nonylphenol degradation by white-rot fungi (44, 45). Isolated extracellular laccases from severalaquatic and terrestrial fungi were shown to catalyze the for-mation of oligomeric coupling products from nonylphenols viaorganic radical intermediates (6, 11). However, the effects oflaccase reactions on the isomeric patterns of t-NP have notbeen assessed to date.

The aim of the present study was to quantify the influence ofextracellular laccase catalysis and intracellular biotransformationon nonylphenol removal rates and on the isomeric composition oft-NP. For this, C. aquatica was used as a model organism. Thederived data were compared to effects of bacteria on nonylphenolisomers reported by other authors (15), and environmental andbiotechnological implications of fungal t-NP biotransformationwere deduced. At the same time we addressed metabolite forma-tion from t-NP and the two major t-NP isomers 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpen-tyl)phenol (NP112) (Fig. 1). This was done to substantiate theapparent differences between fungi and bacteria in the intracel-lular oxidation of t-NP (11, 15).

MATERIALS AND METHODS

Chemicals. All chemicals were of analytical grade (or gradient grade in thecase of chromatography solvents) unless otherwise stated. ABTS [2,2�azinobis(3-ethylbenzthiazolinesulfonic acid)] and vanillic acid were purchased from Sigma-Aldrich (Steinheim, Germany). t-NP (purity 84%) was obtained from Fluka(Neu-Ulm, Germany). All other commercially available chemicals were pur-chased from Merck (Darmstadt, Germany). Two nonylphenol isomers, 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpentyl)phe-nol (NP112), were synthesized via Friedel-Crafts alkylation as describedpreviously (37).

Organism. The source, identification, and maintenance of the AQH C.aquatica de Wild. strain WD(A)-00-1 have been described previously (23).

Biotransformation of nonylphenols in fungal cultures. Liquid culture experi-ments with C. aquatica were conducted in 125-ml Erlenmeyer flasks containing37.5 ml of a 1% (wt/vol) malt extract medium (pH 5.6–5.8), providing cometa-bolic conditions. After inoculation as has been previously described (23), fungalcultures were incubated at 14°C and 120 rpm in the dark for the time periodsindicated in the text. To assess the influence of extracellular laccase on nonyl-phenol removal, a mixture of 50 �M CuSO4 and 1 mM vanillic acid was addedto half of the fungal cultures at culture day 4 to stimulate extracellular laccaseproduction in C. aquatica (23). At culture day 15 t-NP and NP112 were asepti-cally added in methanolic stock solutions to give final concentrations of 75 �M(corresponding to 1% [vol/vol] methanol). This coincided with the onset of thestationary growth phase where substantial levels of extracellular laccase activitywere reached (Fig. 2B and D) (23). To improve the solubility of nonylphenols,0.1% (wt/vol) Tween 80 was also added. At the same time as the addition ofnonylphenols, 0.5 g of cycloheximide liter�1 was added to half of the cultureswhere laccase production had been stimulated as described above and to half ofthe cultures without CuSO4 and vanillic acid. This was done to suppress theformation of degrading enzymes. During further cultivation, upon visual exam-ination no obvious changes in biomass development and fungal morphology werenoticed potentially caused by the addition of cycloheximide. As controls, fungalcultures that had been heat inactivated by autoclaving for 30 min at 121°C priorto nonylphenol addition, as well as fungal cultures omitting nonylphenols, wereused.

Fungal cultures that had received t-NP and NP112 on culture day 15 were usedto determine apparent first-order decay constants (k�) for nonylphenols underthe different conditions used. To determine k� values, error-weighted linearregression of semilogarithmic plots of nonylphenol concentrations versus timewas performed using OriginPro 8G software (OriginLab Corp., Northampton,MA). The k� values were determined from culture days 15 to 21 (from culturedays 15 to 19 for laccase-expressing cultures containing NP112 and cyclohexi-mide). The correlation coefficients were �0.98 (all P values were �0.001) fort-NP-containing cultures with or without laccase induction, �0.97 (P � 0.001) forlaccase-expressing cultures containing NP112, �0.96 (P � 0.002) for NP112-containing cultures without laccase induction, �0.94 (P � 0.005) for laccase-expressing cultures containing t-NP and cycloheximide, and �0.91 (P � 0.03) forlaccase-expressing cultures containing NP112 and cycloheximide. Fungal nonyl-phenol removal was considered as negligible for t-NP- and NP112-containingcycloheximide-treated cultures not displaying laccase activity, since at the end ofexperiments nonylphenol concentrations were not significantly lower than thoseof the respective controls (Fig. 2A and C). Maximal nonylphenol removal rateswere calculated by multiplying the initial nonylphenol concentration with thecorresponding k� value.

Further experiments addressed the biotransformation of the nonylphenol iso-mers NP111 and NP112 added to laccase-lacking fungal cultures at culture day3. This corresponded to the onset of the exponential growth phase, which wasalso used for t-NP addition in our previous study (23). NP111 and NP112 wereaseptically added from methanolic stock solutions at 75 �M, together with 0.1%Tween. Two types of fungal control cultures were prepared as described above.

Laccase-catalyzed removal of t-NP. A laccase-containing concentrated crudeculture supernatant of C. aquatica served as an enzyme source in the experimentsaddressing enzymatic t-NP removal. Laccase production by C. aquatica andconcentration of the cell-free crude culture supernatants were performed asdescribed previously (23). The absence of manganese peroxidase, manganese-independent peroxidase, and lignin peroxidase was verified, as has been reportedearlier (23). Enzymatic reaction mixtures were prepared containing laccase at 0.4U ml�1, 250 �M t-NP added in methanol (final methanol concentration of 1%),and 0.1% Tween 80 in 100 mM sodium citrate buffer (pH 4.0). Assays containingheat-inactivated laccase, prepared by autoclaving at 121°C for 30 min, did notdisplay any laccase activity and served as controls. Enzymatic reaction mixtureswere incubated at 120 rpm and 24°C for 6 days in the dark before analyzing.

FIG. 1. Chemical structures of the nonylphenol isomers NP111 andNP112.

VOL. 75, 2009 BIOTRANSFORMATION OF NONYLPHENOL BY C. AQUATICA 4399


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HPLC analysis of nonylphenols. Concentrations of t-NP and nonylphenolisomers in cell-free supernatants of fungal cultures and enzymatic degradationexperiments were determined by high-performance liquid chromatography(HPLC). Samples were taken at the time points indicated in the text. Samplepreparation, and the Merck-Hitachi HPLC system (Merck-Hitachi, Dusseldorf,Germany) used for the analysis have been described previously (23). Briefly,acetonitrile and distilled water served as eluents as follows (0.5 ml min�1 flowrate, detection at 225 nm): 40% (vol/vol) acetonitrile (2 min), linear increase to90% acetonitrile (8 min), isocratic elution (2 min), linear decrease to 40%acetonitrile (8 min), and final isocratic elution (5 min). External calibration wasused to quantify the nonylphenols.

GC-MS analysis of nonylphenols and biotransformation products. After acid-ification of the harvested fungal cultures to pH 2 with concentrated acetic acid,these were homogenized at 24,000 rpm with a mechanical homogenizer (Ultra-turax; IKA, Heidelberg, Germany) and extracted twice with 15 ml of ethylacetate. The emptied Erlenmeyer flasks were rinsed with additional 15 ml ofethyl acetate. Samples (1 ml) from the enzymatic reaction mixtures were acidi-fied as described above and extracted twice with 1 ml of ethyl acetate. The ethylacetate fractions were combined, dried over anhydrous sodium sulfate, andevaporated to dryness by using a Syncore Pylovap apparatus (Buchi, Flawil,Switzerland). Solvent-free residues were redissolved in 1 ml of n-hexane andanalyzed by gas chromatography-mass spectrometry (GC-MS) using a 6890 gaschromatograph equipped with a 5973 mass sensitive detector (Agilent Technol-ogies, San Jose, CA) and an HP-5 MS fused-silica column (30 m by 0.25 mm[inner diameter], 0.25-�m film thickness; Agilent Technologies) as previouslydescribed (23). Analysis was conducted in full scan mode over an m/z range of 50to 500 atom mass units (amu).

Laccase activity measurements. Extracellular laccase activities in fungal liquidcultures and cell-free concentrated crude culture supernatants were determinedby the oxidation of 3 mM ABTS as has been described previously (23). Enzymeactivities are expressed as units (1 U corresponds to 1 �mol of product formedper min).

RESULTS

Influence of laccase on nonylphenol concentrations in fun-gal cultures. HPLC analysis of culture supernatants was used

to monitor the removal of t-NP and NP112 after their additionto pregrown C. aquatica cultures (Fig. 2). The highest removalrates were observed in fungal cultures that were supplementedwith CuSO4 and vanillic acid. In these cultures, extracellularlaccase activities of �350 (t-NP-containing cultures) and 450(NP112-containing cultures) U/liter were present at the timepoint of nonylphenol addition (Fig. 2B and D). Under suchconditions, nonylphenol concentrations decreased to about 27(t-NP) and 24 �M (NP112) by the end of the experiment (Fig.2A and C). Laccase activities further increased to �800 U/literon culture days 25 (t-NP-containing cultures) and 21 (NP112-containing cultures) and subsequently declined (Fig. 2B andC). Concentrations of t-NP and NP112 of about 52 and 72 �M,respectively, were recovered from heat-inactivated controls(Fig. 2A and C). This corresponds to ca. 69 and 96% of the 75�M initially added. In additional experiments, cycloheximidewas included at the time point of nonylphenol addition. Non-ylphenol removal was clearly less efficient under these condi-tions, and final t-NP and NP112 concentrations were approx-imately 36 and 51 �M, respectively (Fig. 2A and C).Extracellular laccase activities increased for only one more dayto about 500 (t-NP-containing cultures) and 450 (NP112-con-taining cultures) U/liter and then continuously declined (Fig.2B and D). This observation indicates an inactivation over timeof the laccase already present at the time point of cyclohexi-mide addition. Proteolysis, which is often implicated in fungallaccase turnover (36, 54), and the concomitant suppression offurther laccase production by the cycloheximide might explainthe observed decrease in laccase activities.

In order to exclude the effect of extracellular laccase onnonylphenol removal, the laccase-inducing mixture of CuSO4

FIG. 2. Concentrations of t-NP (A) and NP112 (C) and extracellular laccase activities in t-NP-containing (B) and NP112-containing (D) C.aquatica cultures that had received nonylphenols on culture day 15. Laccase-induced cultures (F), laccase-induced cultures containing cyclohex-imide (E), laccase-lacking cultures (f), laccase-lacking cultures with cycloheximide (�), and heat-inactivated control cultures (�). The datarepresent means the standard deviations from triplicate cultures. Final nonylphenol concentrations (culture day 27) were significantly lower (P �0.02) than nonylphenol concentrations in the corresponding controls according to the Student t test, except those in cycloheximide-treated cultureswithout extracellular laccase activity.

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and vanillic acid was omitted. Under these conditions, onlynegligible extracellular laccase activities were detected (Fig. 2Band D). Final t-NP and NP112 concentrations of about 41 and61 �M were obtained in fungal cultures (Fig. 2A and C). Thisindicates that fungal cultures not exhibiting substantial laccaseactivities are less efficient in nonylphenol removal than culturesshowing high extracellular laccase activities. The nonylphenoldepletion observed in laccase-lacking cultures can be attrib-uted to as-yet-unknown intracellular enzymes acting on nonyl-phenol (23, 34). Cycloheximide was also applied under condi-tions where essentially no extracellular laccase activities werepresent. Under such conditions, no biological nonylphenol re-moval occurred. This was evident from the final nonylphenolconcentrations, which were not significantly lower than thoseof the corresponding controls (Fig. 2A and C).

To quantify the effects of laccase on the initial nonylphenolremoval rates, first-order kinetics were applied to time coursesof nonylphenol concentrations in fungal cultures (Table 1).Satisfactory fits were obtained during the initial 4 to 6 daysafter nonylphenol addition (see Materials and Methods). Afterthis time, decreasing physiological activity due to increasingnutrient depletion might have been responsible for the stag-nation in nonylphenol removal (Fig. 2A and C). In laccase-induced cultures, the maximal t-NP removal rate was �1.6-foldhigher than in laccase-lacking cultures without cycloheximideadded (Table 1). Cycloheximide addition lowered the maximalt-NP removal rate in laccase-induced cultures by ca. 40% andcompletely inhibited t-NP removal in cultures without laccase.An even more pronounced effect of laccase on nonylphenolremoval was observed in experiments with NP112. In laccase-induced cultures, the maximal removal rate of this single iso-mer was �2.4-fold higher than in cultures devoid of laccase(Table 1). Cycloheximide reduced the maximal NP112 removal

rate in laccase-induced cultures by ca. 50%, whereas it com-pletely inhibited NP112 removal in cultures without laccase.The maximal removal rate of t-NP observed with laccase-in-duced cultures in the presence of cycloheximide was essentiallycomparable to that obtained with laccase-lacking cultures inthe absence of cycloheximide (Table 1). The same effect wasobserved for the respective cultures containing NP112 (Table1). Furthermore, an inhibition of the formation of intracellularenzymes acting on t-NP and NP112 by cycloheximide is indi-cated. Intracellular degrading enzymes were obviously inducedby the nonylphenols (Fig. 2A and C and Table 1).

Influence of laccase on removal patterns of isomer groups oft-NP. C. aquatica cultures, which had received t-NP on cultureday 15, were harvested and extracted with ethyl acetate after 27days of cultivation, i.e., when nonylphenol removal had essen-tially become stagnant (Fig. 2A and C). GC-MS analysis of theextracts showed 12 peaks representing different groups ofnonyl chain-branched t-NP isomers and yielding individualmass spectra (Table 2). Mass spectra of all of the separatedt-NP constituents indicate common structural features con-cerning the branching positions in the nonyl chains as has beendescribed previously (23, 55). The resolved groups of t-NPisomers appeared at different concentrations as demonstratedby the total peak areas of ions corresponding to separatedcompounds over an m/z range of 50 to 500. These are shownfor heat-inactivated fungal cultures (controls) in Fig. 3A. Boththe mass spectra (Table 2) and the concentration profile of theseparated t-NP constituents (Fig. 3A) agree with those of 12t-NP constituents separated under similar GC-MS conditionsin a previous study (24). This allows the unambiguous alloca-tion of the peaks observed in the t-NP mixture from thepresent study to the corresponding t-NP isomer peaks and theidentified component isomers as has been described by others(15, 24) (Table 3).

The percentage concentrations, relative to the respectiveconcentrations of resolved t-NP constituents in heat-inacti-vated controls, of individual t-NP isomers peaks in the differ-ently treated fungal cultures are depicted in Fig. 3B, D, and E.In fungal cultures where laccase production had been inducedon culture day 4 and further enzyme formation was suppressedby cycloheximide added together with t-NP on culture day 15(Fig. 2A and B), an essentially uniform removal of all sepa-rated t-NP isomer groups was observed (Fig. 3B). The concen-trations of almost all of the t-NP constituents decreased by ca.45 to 50%, whereas isomer peak 7 decreased by ca. 35%. Sincecycloheximide obviously prevented intracellular t-NP biotrans-formation (Fig. 2A), this removal pattern mainly reflects theinfluence of laccase on t-NP. A qualitatively comparable effecton the removal pattern of resolved t-NP isomer groups wasobserved in enzymatic reaction mixtures containing a crudelaccase preparation from C. aquatica (Fig. 3C). Here, 21.0% 5.5% (means the standard deviations from triplicate exper-iments) of the t-NP sum concentration of 250 �M initiallyapplied was removed during 6 days of incubation. Taken to-gether, these results indicate that laccase oxidizes the isomerscontained in t-NP in an unspecific manner.

In fungal cultures where laccase was induced but cyclohex-imide was omitted (Fig. 2A and B), a much more specificremoval of the t-NP isomers was detected (Fig. 3D), comparedto cultures where cycloheximide had prevented intracellular

TABLE 1. Apparent first-order decay constants and maximalnonylphenol removal rates observed in differentially

treated C. aquatica cultures upon nonylphenoladdition on culture day 15

Nonylphenol andculture conditions k� (day�1)a

Maximalremoval rate(�mol liter�1

day�1)b

t-NPLaccase-induced cultures 0.139 0.036 8.54Laccase-induced cultures �

cycloheximide0.091 0.015 5.32

Laccase-lacking cultures 0.083 0.006 5.44Laccase-lacking cultures �

cycloheximideNRc NR

NP112Laccase-induced cultures 0.171 0.029 13.97Laccase-induced cultures �

cycloheximide0.089 0.023 7.07

Laccase-lacking cultures 0.070 0.009 5.84Laccase-lacking cultures �

cycloheximideNR NR

a Values standard errors were determined upon linear regression of semi-logarithmic plots of nonylphenol concentrations versus time.

b Maximal removal rates were calculated by multiplying the k� value by thecorresponding initial nonylphenol concentration according to first-order removalkinetics.

c NR, no removal.



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bioconversion (Fig. 3B). These differences in the degradationpatterns obviously reflect the concomitant contribution of in-tracellular activities to t-NP removal under conditions wherelaccase is also present. Evidence for a clearly more selectiveattack on individual t-NP isomers by intracellular enzymes,compared to laccase oxidation, was obtained from fungal cul-tures without laccase production where only intracellular ac-tivities were present (Fig. 3E). Under these conditions, theconcentrations of isomer peaks 3, 10, and 11 decreased by ca.69 to 73%, isomer peaks 2 and 4 decreased by ca. 55 to 57%,and the majority of the remaining isomer peaks decreased byca. 30 to 46%.

Formation of biotransformation products of t-NP. To detectmetabolites resulting from the intracellular attack on t-NP,ethyl acetate extracts of t-NP-containing fungal cultures devoidof extracellular laccase activities were analyzed by GC-MS. Inactive C. aquatica cultures that had received t-NP on cultureday 15 and were further incubated until t-NP removal hadbecome stagnant (culture day 27, Fig. 2A), the metaboliteslisted in Table 4 were detected. These compounds were absentin the corresponding fungal control cultures, as well as inparent t-NP. Therefore, these are indicative of the biotrans-formation of t-NP by intracellular enzymes. The mass spectraof all of these products contained the characteristic hydroxyltropylium ion at m/z 107 (55), which is also present in isomersof parent t-NP (Table 2). This proves that the biotransforma-tion reactions did not occur at the aromatic ring of t-NP (23).Various metabolites with molecular ions at m/z 236 (Table 4),representing nonyl chain-monohydroxylated t-NP isomers,have already been described for C. aquatica (23, 34). Othert-NP metabolites such as those with molecular ions at m/z 218,234, and 278 (Table 4) have not been detected before in fungi.The most intense fragment ions in the mass spectra of metab-olites were observed at m/z 149 and m/z 135 (Table 4), sug-gesting �-ethyl, �-methyl, and �,�-dimethyl configurations, re-spectively (33). The simultaneous detection of strong fragmentions at both m/z 149 and m/z 135 (metabolite peaks B-2, B-4,

and C-1; Table 4), points to coelution of several metabolites(Table 4). For metabolite A-1, the fragment ion at m/z 149(elimination of -C5H9) indicates desaturation of a carbon bondin the C5 alkyl residue attached to the quaternary �-carbon(Fig. 4). A desaturation in the C6 alkyl moiety connected to the�-carbon of metabolite A-2 is supported by fragment ions atm/z 163 and 135 (Table 4). Compound C-1 with a molecularion at m/z 234 (Table 4) suggests further oxidation into alde-hyde or keto derivatives of t-NP isomers with monohydroxy-lated nonyl chains. Metabolite D-1 corresponds to metaboliteD-2 (Table 4) observed upon application of NP112 to C.aquatica cultures (see below). A possible structure and anexplanatory mass fragmentation pathway is suggested in Fig. 4.

No t-NP metabolites were detected upon GC-MS analysis ofethyl acetate extracts of laccase reaction mixtures. The forma-tion of oxidative coupling products from t-NP in the range ofdi- to pentamers has consistently been described for differentfungal laccases in previous studies, where oligomeric com-pounds were separated from enzymatic reaction mixtures uponprecipitation (6, 23). The detection of oligomeric couplingproducts from t-NP was not expected within the present studysince (i) the applied ethyl acetate extraction procedure likelyyields insufficient product amounts with higher molecularmasses, and (ii) tri- to pentamers would be inaccessible usingthe GC-MS method applied (covered mass range of 50 to 500amu).

Formation of biotransformation products of NP112. NP112has been described as accounting for up to ca. 13% of theisomers contained in t-NP (37). In addition to the para-substi-tuted isomer, representing the major constituent, the NP112preparation used in the present study contained ca. 11% of thecorresponding ortho-isomer (Table 2). Similar proportionswith respect to the para- and ortho-isomers of NP112 havebeen reported previously (33). Due to its retention time andmass spectrum, para-substituted NP112 contributed to the iso-mer peak 3 in the GC-MS chromatogram of parent t-NP (Ta-ble 2). Coelution of further t-NP isomers as has also been

TABLE 2. Mass spectral data and GC-MS retention times of the nonylphenol preparations used within the present study

Nonylphenol Isomer peak GC retentiontime (min) m/z of prominent ions (relative abundance %�)

t-NP 1 15.21 220 (M�, 9), 177 (20), 163 (48), 149 (4), 121 (100), 135 (12), 107 (51)2 15.29 220 (M�, 5), 135 (100), 121 (7), 107 (11)3 15.37 220 (M�, 19), 191 (36), 149 (86), 135 (100), 121 (51), 107 (66)4 15.42 220 (M�, 8), 191 (8), 149 (39), 135 (100), 121 (32), 107 (28)5 15.47 220 (M�, 8), 191 (19), 163 (2), 149 (100), 135 (19), 121 (76), 107 (46)6 15.54 220 (M�, 6), 177 (18), 163 (22), 135 (100), 121 (35), 107 (43)7 15.58 220 (M�, 4), 177 (2), 163 (100), 135 (16), 121 (80), 107 (73)8 15.62 220 (M�, 3), 177 (23), 163 (3), 149 (100), 135 (7), 121 (36), 107 (54)9 15.65 220 (M�, 5), 177 (8), 163 (100), 149 (37), 135 (53), 121 (93), 107 (93)10 15.69 220 (M�, 2), 135 (100), 121 (3), 107 (12)11 15.73 220 (M�, 12), 191 (42), 163 (10), 149 (100), 135 (61), 121 (49), 107 (95)12 15.77 220 (M�, 3), 149 (100), 135 (11), 121 (11), 107 (36)

NP112 ortho-Isomer 14.83 220 (M�, 12), 191 (21), 149 (60), 135 (10), 121 (60), 107 (100)para-Isomer 15.39 220 (M�, 20), 191 (58), 149 (100), 135 (17), 121 (74), 107 (85)

NP111 ortho-Diastereomer a 14.79 220 (M�, 11), 191 (11), 149 (68), 135 (5), 121 (100), 107 (88)ortho-Diastereomer b 14.87 220 (M�, 10), 191 (11), 149 (62), 135 (4), 121 (100), 107 (81)para-Diastereomer a 15.42 220 (M�, 10), 191 (20), 149 (100), 135 (10), 121 (77), 107 (50)para-Diastereomer b 15.47 220 (M�, 11), 191 (24), 149 (100), 135 (9), 121 (80), 107 (51)



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observed in previous studies (24, 37) resulted in partly differingmass spectra. The assignment of NP112 to the GC-MS peak 3(Table 3) is in accordance with results of a previous study (24).

Active C. aquatica cultures were supplemented with NP112on culture day 15 and extracted with ethyl acetate after a totalcultivation time of 27 days (Fig. 2C). In additional experimentsNP112 was added to C. aquatica cultures on culture day 3,which then were incubated for another 28 days prior to ethylacetate extraction. This incubation period was chosen since itcorresponds to the one used in our pervious study, where t-NPremoval by C. aquatica investigated under otherwise compara-ble conditions was found to become stagnant (23). In culturesthat had received NP112 on culture day 3, HPLC analysis ofthe culture supernatants yielded a final concentration of thecompound of 16 0.0 �M (mean the standard deviationfrom triplicate cultures). This corresponds to 21.3% of theamount initially added. A concentration of 54.0 9.6 �M

(72.0% of the initial amount) was recovered from heat-inacti-vated controls. GC-MS analysis of ethyl acetate extracts ofboth types of cultures yielded the compounds listed in Table 4.These were not found in parent NP112 and in the correspond-ing fungal control cultures. Comparable retention times andmass spectral data indicate that compounds A-3, A-4, B5, andB6 are identical to metabolites A-5, A-6, B7, and B-8, respec-tively (Table 4). In accordance with the similar GC retentiontimes and mass spectral data, the NP112 metabolites A-4 (�A-6), B-5 (� B-7), B-6 (� B-8), and D-2 can also be assignedto compounds detected during biotransformation of t-NP(peaks A-1, B-2, B-4, and D-1, respectively). This observationimplies that NP112 is also attacked when a component of theisomeric mixture of t-NP and possibly reflects the high contentof NP112 in t-NP (37). Mass spectra of all NP112 metabolitesfeature diagnostic fragment ions indicative of an attack exclu-sively at the alkyl moiety, thus confirming the results obtained

FIG. 3. Absolute GC-MS peak areas (mega-arbitrary units [MAU]) of separated t-NP isomer groups in heat-inactivated fungal control cultures(A) and percentage concentrations (in relation to the respective concentrations of the corresponding t-NP constituents in heat-inactivated controls,which represent 100%) of separated t-NP constituents in laccase-induced C. aquatica cultures containing cycloheximide (B), reaction mixturescontaining a laccase preparation from C. aquatica (C), laccase-induced C. aquatica cultures without cycloheximide (D), and C. aquatica culturesdevoid of extracellular laccase activity (E). The labeling of t-NP isomer peaks relates to that in Tables 2 and 3. Active fungal cultures andcorresponding heat-inactivated controls received t-NP on culture day 15 and were analyzed on culture day 27. Laccase-containing reaction mixtureswere analyzed after 6 days of incubation in the presence of t-NP. Calculations were based on respective total peak areas of ions corresponding toseparated t-NP constituents over an m/z range of 50 to 500. Bars and error bars represent means the standard deviations for triplicate culturesand enzymatic reaction mixtures.



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with t-NP (Table 4) (23). A mass loss difference of 29 amu(elimination of -C2H5 from the �-carbon) between the respec-tive molecular ions and corresponding fragment ions was ob-served for all NP112 metabolites (Table 4). This excludes thepossibility of structural alterations in the ethyl substituent ofthe �-carbon (Fig. 4). The mass spectral data of metabolitesA-3 (� A-5) and A-4 (� A-6) (Table 4) imply desaturation of

a carbon bond along the C5 alkyl moiety attached to the �-car-bon (Fig. 4). Different possible positions of the unsaturatedcarbon bond would explain the detection of more than oneNP112 metabolite with a molecular ion at m/z 218. The massspectra of metabolites B-5 (� B-7) and B-6 (� B-8) (molecularion at m/z 236, respectively) indicate monohydroxylation of thenonyl chain, which was also observed for t-NP (Table 4) (23).The most intense fragment ion at m/z 149 (loss of -C5H11O)observed in the mass spectra of compounds B-5 (� B-7) andB-6 (� B-8) (Table 4), excludes the possibility of a hydroxyla-tion of the methyl substituent of the �-carbon. Therefore,hydroxylation can only have taken place at the C5 alkyl residueattached to the �-carbon, and two possible structures areshown in Fig. 4. Metabolite E-1 can be explained by furtheroxidation of a hydroxylated precursor to a carboxylic acid de-rivative. A proposed structure and an explanatory mass frag-mentation pathway is shown in Fig. 4. The molecular ion at m/z278 and the accompanying fragment ions in the mass spectrumof metabolite D-2 may indicate methylation of a carboxylgroup in a precursor, such as metabolite E-1, leading to theformation of a methyl ester (Fig. 4).

Formation of biotransformation products of NP111. NP111has been reported to account for up to ca. 20% of the isomerscontained in t-NP (37). The NP111 preparation used for thebiotransformation experiments with C. aquatica cultures con-sisted of a pair of para-substituted diastereomers (Table 2) asthe major constituents. These were present at nearly identicalconcentrations as has been previously described (33). In addi-tion, NP111 contained ca. 5% of the corresponding ortho-isomers, again similar to previously published data (33). Ac-cording to its retention time and mass spectral characteristics,

TABLE 3. Allocation of GC-MS peaks observed for the t-NPmixture within the present study to the corresponding

t-NP isomer peaks and the identified componentisomers as described in the literature

Isomerpeaka

Corresponding GC-MSpeak(s) of the t-NP

mixture in:Isomer(s) contributing to t-NP peak(s)

according to references 15 and 22(mass spectrometric group)b

Reference22

Reference15

1 1 1 NP194 (5)2 2 7 NP36 (1)3 3 9, 13, 14 NP38 (1), NP112 (2), NP128 (1)4 4 16, 17 NP37 (1), NP111a* (2)5 5 19 NP111b* (2)6 6 24, 26 NP119 (1), NP152 (5)7 7 27 NP193a* (4)8 8 29 NP110a* (3)9 9 30 NP193b* (4)10 10 31 NP35 (1)11 11 32, 33 NP9 (1), NP65 (2)12 12 40 NP110b* (3)

a Labeling of t-NP isomer peaks refers to that of Table 2.b The numbering system for nonylphenol isomers described in reference 18

was applied. Assignment to mass spectrometric groups concerning the substitu-tion pattern of the quaternary �-carbon was done according to reference 15. �,NP110, NP111, and NP193 each consist of a pair of diastereomers.

TABLE 4. Mass spectral data and GC-MS retention times of biotransformation metabolites of t-NP, NP112, and NP111 detected inC. aquatica cultures devoid of extracellular laccase activities

Parent nonylphenol andday of additiona

Metabolitepeak no.

Relative GC retentiontime (min)b m/z of prominent ions (relative abundance %�)

t-NP, day 15* A-1 102.73 218 (M�, 6), 189 (9), 149 (100), 133 (11), 121 (12), 107 (65)A-2 103.36 218 (M�, 16), 163 (6), 147 (9), 135 (100), 124 (7), 121 (7), 107 (33)B-1 106.09 236 (M�, 5), 147 (5), 135 (100), 121 (5), 107 (12)B-2 106.28 236 (M�, 10), 207 (11), 189 (45), 149 (100), 135 (27), 121 (12), 107 (46)C-1 106.98 234 (M�, 19), 205 (44), 187 (53), 172 (13), 149 (100), 135 (58), 121 (26), 107 (57)B-3 107.04 236 (M�, 5), 191 (4), 135 (100), 121 (9), 107 (15)B-4 111.98 236 (M�, 8), 207 (17), 189 (20), 149 (100), 135 (33), 121 (16), 107 (51)D-1 116.23 278 (M�, 5), 249 (21), 189 (14), 149 (100), 133 (6), 121 (12), 107 (37)

NP112, day 15* A-3 99.37 218 (M�, 14), 189 (36), 149 (100), 133 (11), 121 (22), 107 (77)A-4 102.85 218 (M�, 6), 189 (10), 149 (100), 133 (10), 121 (14), 107 (85)B-5 106.59 236 (M�, 10), 207 (12), 189 (47), 149 (100), 133 (9), 121 (9), 107 (42)B-6 112.30 236 (M�, 8), 207 (17), 189 (21), 149 (100), 133 (8), 121 (17), 107 (46)D-2 116.23 278 (M�, 7), 249 (22), 189 (15), 149 (100), 134 (8), 121 (11), 107 (37)

NP112, day 3† A-5 99.49 218 (M�, 12), 189 (36), 149 (100), 133 (11), 121 (20), 107 (73)A-6 103.17 218 (M�, 5), 189 (9), 149 (100), 133 (13), 121 (15), 107 (99)B-7 106.85 236 (M�, 8), 207 (11), 189 (42), 149 (100), 134 (9), 133 (8), 121 (10), 107 (46)B-8 112.87 236 (M�, 5), 207 (11), 189 (24), 149 (100), 134 (6), 133 (6), 121 (9), 107 (21)E-1 114.46 264 (M�, 6), 235 (26), 189 (5), 175 (5), 149 (100), 134 (9), 133 (8), 121 (19), 107 (52)

NP111, day 3† C-2 106.34 234 (M�, 18), 205 (35), 187 (39), 149 (100), 121 (16), 107 (5)C-3 106.98 234 (M�, 13), 205 (27), 187 (38), 149 (100), 121 (10), 107 (34)

a �, analyzed after a total cultivation time of 27 days; †, analyzed after a total cultivation time of 31 days.b Retention times are expressed in relation to that of the t-NP isomer peak 12 (15.77 min, Table 2) used as a marker for comparison, due to slightly shifted retention

times in different separations.



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the para-diastereomer NP111a contributed to the isomer peak4 in the GC-MS chromatogram of parent t-NP (Table 2).Coelution of further t-NP isomers has also been reported pre-viously (24, 37), and resulted in partly differing mass spectra.Due to its GC retention time and mass spectrum, the para-diastereomer NP111b can be assigned to the isomer peak 5 oft-NP (Table 2). The assignment of NP111a and NP111b to theGC-MS peaks 4 and 5, respectively, is consistent with previ-ously reported data (Table 3) (24).

The formation of metabolites resulting from the biotrans-formation of NP111 was followed with C. aquatica cultures thathad received the parent compound on culture day 3. Afterincubation of the cultures for another 28 days, HPLC analysisof the culture supernatants yielded a final NP111 concentra-tion of 21.5 2.9 �M. This corresponded to 28.7% of the

amount initially added. A concentration of 60.6 1.8 �M(80.8% of the initial amount) was recovered from heat-inacti-vated controls. The compounds C-2 and C-3, present in theactive fungal cultures (Table 4), were absent in parent NP111,and also in the corresponding fungal control cultures. Due toits comparable GC retention time and a similar mass spectrum,the compound C-3 can be assigned to metabolite peak C-1arising from t-NP conversion. This is despite the coelution ofmore than one compound contributing to metabolite peak C-1(indicated by strong signals at both m/z 149 and 135, Table 4).This observation implies that NP111 is also converted whencontained in t-NP, which is in agreement with its reported highconcentration in the technical isomer mixture (37). As wasobserved for the t-NP and NP112 metabolites, diagnostic frag-ment ions contained in the mass spectra of NP111 biotrans-formation products were indicative of structural modificationsoccurring exclusively in the nonyl chains (23). The molecularions of compounds C-2 and C-3 (both at m/z 234) suggestaldehyde or keto derivatives resulting from further oxidation ofnonyl chain-hydroxylated precursors, as already stated for thet-NP metabolite C-1. However, the several possibilities forinterpreting of the mass spectra of metabolites C-2 and C-3 donot allow the structures to be elucidated in more detail. Twoketo derivatives, 3-(4-hydroxyphenyl)-3,5-dimethylheptan-4-one and 5-(4-hydroxyphenyl)-3,5-dimethylheptan-2-one, haverecently been proposed as metabolites arising from treatmentof NP111 in a membrane bioreactor (9). Similar to the twodiastereomers of NP111, metabolites C-2 and C-3 appeared atnearly identical concentrations, as deduced from the total peakareas of the corresponding ions over an m/z range of 50 to 500.The mass spectra of metabolites C-2 and C-3 are also verysimilar (Table 4). Taking all together, this may indicate thatthese compounds represent diastereomers arising from thesimultaneous conversion of the two diastereomers of NP111.This assumption is supported by the observation that the con-centration ratio of NP111a and NP111b did not change duringbiotransformation of these isomers by fungal cultures.

Contribution of detected t-NP metabolites to the overallt-NP biotransformation process. A precise determination oft-NP metabolite concentrations was impeded by the unavail-ability of appropriate reference compounds. In order to pro-vide an order of magnitude estimate of concentrations of me-tabolites detected during t-NP biotransformation in theabsence of laccase activity, the relative GC abundances of t-NPmetabolites are shown in Table 5. These estimates are biasedby different intensities of the ions in the mass spectra of theseparated t-NP constituents (Table 2) and t-NP metabolites(Table 4) and also potentially by the coelution of more thanone compound contributing to the t-NP metabolite peaks B-2,B-4, and C-1 (Table 4). Nevertheless, they should allow arough estimate of the extent to which the metabolites reflectthe overall t-NP removal process. A sum GC abundance of ca.44% for all detected metabolites (Table 5) suggests that a largeamount of the t-NP constituents were converted into thesecompounds. Other t-NP metabolites which were possibly pro-duced at only very low concentrations would not have allowedfor their unambiguous detection. Furthermore, polar sugar orsulfate conjugates of nonylphenols may potentially have beenproduced from t-NP, as has been reported for plants (4, 41)and animals (28, 58). These would not be accessible with the

FIG. 4. Examples for possible structures and suggested main frag-mentation pathways (dashed lines where precursor ions other thanmolecular ions are suggested) of metabolites resulting from intracel-lular biotransformation of t-NP and NP112, as based on GC-MS anal-ysis. The corresponding m/z values of molecular ions (M�) of metab-olites and further prominent ions are also shown. The metabolitenumbering (in parentheses) refers to Table 4.



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analytical procedure applied within the present study. Duringpreliminary experiments with [ring-U-14C]NP111 and [ring-U-14C]NP112 applied to laccase-lacking C. aquatica cultures,�5% of the radioactivity initially added was found to be asso-ciated with active fungal biomass (C. Martin, P. F. X. Corvini,and D. Schlosser, unpublished data).

DISCUSSION

Within the present study, we have quantified the effects ofextracellular laccase and intracellular biotransformation reac-tions on the isomer-specific removal of the xenestrogen t-NPby C. aquatica. In addition to as-yet-unknown intracellularenzymes, extracellular laccase largely accounts for the removalof nonylphenol isomers, leading to a considerable increase inbiotransformation rates when laccase oxidation is operative(Fig. 2 and 3 and Table 1). For both t-NP and NP112, themaximal removal rates observed under conditions where lac-case was present and intracellular reactions were suppressed(laccase-induced cultures with cycloheximide added) were es-sentially comparable to those obtained when intracellular non-ylphenol attack was active and laccase was absent (laccase-lacking cultures without cycloheximide), respectively (Table 1).These results illustrate the importance of both processes forthe entire biotransformation mechanism and indicate a nearlyequal contribution of laccase oxidation and intracellular reac-tions to nonylphenol removal. It remains uncertain whetherthe cycloheximide effects observed within our study were dueto a translational inhibition of the enzyme protein biosynthesis.An inhibition of the de novo synthesis of laccase proteincaused by 25 mg of cycloheximide liter�1 was implicated in

Aspergillus nidulans (29). In contrast, low concentrations ofcycloheximide were reported to induce laccase expression inother fungi. In the yeast Neurospora crassa, transcriptionalactivation of the laccase gene upon cycloheximide applied at0.8 mg liter�1 is mediated by the cross-pathway control genecpc-1 (20). Considering the high cycloheximide concentrationof 0.5 g liter�1 applied within our study, a carbon- or nitrogen-based repression of enzyme gene transcription may also berelevant. The repression of the transcription of fungal laccasegenes by glucose has been reported previously (18).

Laccase-catalyzed t-NP oxidation leads to the essentiallyuniform removal of the resolved groups of t-NP isomers con-sistently observed with laccase-expressing fungal cultures in thepresence of cycloheximide and reaction mixtures containingisolated laccase (Fig. 3B and C). A quite unspecific oxidationof the isomers contained in t-NP can be explained by the factthat laccases attack the phenolic OH group, whereby branch-ing of the nonyl chains located in the p-position should be lessimportant. This assumption is supported by the relatively smallsteric hindrances of bulky p-substituents reported for the lac-case-catalyzed oxidations of phenols other than nonylphenol(56). The unspecific oxidation of t-NP isomers by laccase dif-fers remarkably from bacterial ipso-hydroxylation, where thedegradation rates of individual t-NP isomers largely depend onthe isomeric structure (15). In Sphingobium xenophagum Bay-ram, certain isomers belonging to a mass spectrometric isomergroup with an estrogenic potency higher than that of the t-NPmixture were less efficiently degraded than others showing aweak estrogenic potential, thereby altering the isomeric com-position of remaining t-NP and leading to the enrichment ofpotentially more estrogenic isomers. Bacterial ipso-hydroxyla-tion thus may lead to an increase in the specific estrogenicity ofaging material in environmental compartments (15). Such po-tentially hazardous effects would not be expected if laccaseoxidation would be the only biochemical reaction convertingt-NP constituents. For instance, the concentration of the iso-mer peak 6 containing NP119 (Table 3), which has been re-ported in a previous study to be the most estrogenic compoundamong the 13 t-NP isomers assessed (isomer 3E22NP in ref-erence 24), was decreased to essentially the same extent as theconcentrations of other isomer peaks upon laccase oxidation(Fig. 3B and C). Laccase has repeatedly been suggested as abiocatalyst for use in enzyme bioreactors, in order to eliminatenonylphenol and also other endocrine disrupting chemicals incontaminated media (7, 8, 50). The oxidation of t-NP and alsothat of 4-n-nonylphenol containing a linear nonyl chain (11) byvarious fungal laccases led to the formation of oligomeric cou-pling products in the di- up to pentamer range (6, 13, 23) andto the disappearance of the estrogenic activity of the parentcompound(s) (6, 38, 51).

Laccase catalysis may be relevant for fungal t-NP removal innatural aquatic environments, although the clearly more selec-tive elimination of resolved t-NP constituents by intracellularreactions (Fig. 3E) also observed when laccase was operative(Fig. 3D), indicates the simultaneous influence of laccase andintracellular processes on the isomeric composition of t-NP.Various aromatic compounds, extracts of lignocellulosic sub-strates, and plant homogenates are known to enhance laccaseformation in both asco- and basidiomycetes (30, 40, 46).Hence, fungi growing on lignocellulose-containing plant detri-

TABLE 5. Relative GC abundances of biotransformationmetabolites of t-NP detected in laccase-lacking

C. aquatica cultures after a total cultivationtime of 27 days

Metabolitepeak no.a

Relative GCabundance (%)b

A-1............................................................................................. 3.1A-2............................................................................................. 4.8B-1 .............................................................................................10.6B-2 .............................................................................................10.3C-1 ............................................................................................. 2.7B-3 ............................................................................................. 2.3B-4 ............................................................................................. 6.8D-1............................................................................................. 3.2

Sum............................................................................................43.8

a Metabolite labeling refers to that of Table 4. Metabolites A-1, B-2, B-4, andD-1 presumably arise from NP112, and therefore the relative GC abundanceswere based on the NP112-containing isomer peak 3. The mass spectra of me-tabolites A-2, B-1, and B-3 indicate an �,�-dimethyl configuration (most intensefragment ions at m/z 135 Table 4�); therefore, these metabolites can arise fromany t-NP constituent containing isomer(s) with this configuration. Hence, therelative GC abundances were based on the average of the �,�-dimethyl isomer-containing peaks 2, 3, 4, 6, 10, and 11 (Tables 2 and 3). Metabolite C-1 presum-ably arises from NP111, and therefore its relative GC abundance was based onthe average of the NP111-containing isomer peaks 4 and 5.

b That is, the integrated areas of the metabolite peaks in the ion trace chro-matograms at m/z 107 relative to those representing the respective amounts ofthe presumed parent compounds removed from fungal cultures between cultureday 15 (t-NP addition) and 27 (end of the experiment). An m/z of 107 was chosensince the corresponding fragment ions appeared at quite high abundances in themass spectra of both t-NP metabolites (Table 4) and separated isomer peaks ofparent t-NP (Table 2).



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tus in aquatic environments would be expected to producelaccase under such natural conditions. In C. aquatica, a mixtureof the plant-related aromatic compound vanillic acid andthe well-known laccase inducer CuSO4 (30, 46) stimulates lac-case excretion, whereas t-NP does not increase extracellularlaccase activities (Fig. 2) (23, 47). In contrast, t-NP and 4-n-NPhave been shown to induce laccase production in the white-rotbasidiomycete Trametes versicolor (26, 27, 35).

The removal pattern of t-NP isomer groups observed in theabsence of laccase activity (Fig. 3E) indicates that still-un-known intracellular enzymes oxidize individual t-NP isomers ina much more selective manner than laccase (Fig. 3B and C).This leads to alterations in the isomeric composition of re-maining t-NP, as has also been reported for S. xenophagumBayram (15). In this organism, which initiates nonylphenoldegradation via ipso-hydroxylation (C4-position of the aro-matic ring), a strong correlation between the substitution pat-tern of the adjacent quaternary �-carbon and the transforma-tion of t-NP isomers was observed (15). Generally, degradationwas more effective when the �-position was less bulky, and thefollowing ranking was established (the assignment of t-NP iso-mers with relevance for the present study to five of six massspectrometric groups, which were used to classify the substitu-tion pattern of the quaternary �-carbon in reference 15, isshown in Table 3): group 1 � group 2 � group 3 � group 5 �group 6 (not relevant for the present study) � group 4 isomers.Our observation that all isomers of groups 3, 4, and 5 can beattributed to those isomer peaks less efficiently degraded by C.aquatica (Fig. 3E and Table 3) resembles results obtained withS. xenophagum Bayram (15). However, a correlation betweenthe bulkiness at the �-carbon of group 3, 4, and 5 isomers andthe removal rates of corresponding isomer peaks was not ob-served (Fig. 3E), and hence a degradation ranking could not beestablished. In particular isomers belonging to group 4 havebeen shown to be clearly more estrogenic than the complext-NP mixture (15). Furthermore, isomer peak 6 containing thehighly estrogenic NP119 (24) was clearly less efficiently re-moved than certain other separated t-NP constituents (Fig.3E). The conclusion that the isomer-specific degradation oft-NP can lead to the enrichment of more estrogenic isomers inthe parent compound mixture which was previously drawn forS. xenophagum Bayram (15), therefore also applies to C.aquatica. This is true even under conditions where both intra-cellular biotransformation and laccase catalysis are operative(Fig. 3D). However, the substitution pattern of the quaternary�-carbon seems to be less important for the intracellular trans-formation of t-NP isomers by C. aquatica. For instance, group1 and group 2 isomers can also be found in t-NP constituentsshowing either intermediate degradation (isomer peaks 2 and4) or pronounced recalcitrance (isomer peaks 5 and 6). Isomerpeak 5 (containing the group 2 diastereomer NP111b) wasremoved to an extent comparable to that of peaks 1 (group 5isomer), 6 (group 1 and 5 isomers), 7 (group 4 diastereomer),and 9 (group 4 diastereomer) (Fig. 3E and Table 3). In accor-dance with our previous study (23), the mass spectral data ofall nonylphenol biotransformation metabolites (Table 4) showthat reactions catalyzed by intracellular enzymes exclusivelyinvolve the differently branched nonyl chains and not the aro-matic ring. Our preliminary observation that C. aquatica didnot release 14CO2 from [ring-U-14C]NP111 and [ring-U-

14C]NP112 (Martin et al., unpublished) is in agreement withbiotransformation reactions occurring at the nonyl chains.Fungal oxidation of the nonyl chains of t-NP isomers differsfrom the ipso-hydroxylation used by the bacterial genera Sphin-gomonas and Sphingobium. This can explain the observed dif-ferences between C. aquatica and S. xenophagum Bayram inthe efficiency of removing individual t-NP constituents.

In C. aquatica, intracellular reactions modifying nonyl chainsof t-NP isomers include primary hydroxylation, further oxida-tion into aldehyde or keto compounds, and the formation ofcarboxylic acid derivatives (Fig. 4 and Table 4). Similarly, thebiotransformation of NP111 by plant cell cultures led to thedetection of several side chain-hydroxylated compounds andone side chain-carboxylated metabolite (41). In C. aquatica,carboxylic acid compounds may subsequently undergo methyl-ation to form methyl esters (Fig. 4). In another AQH, Heliscuslugdunensis, methylation of xenobiotic compounds has beendemonstrated for 1-naphthol (2). Desaturation as implicatedfor the nonyl chains of certain metabolites of t-NP and NP112(Fig. 4 and Table 4) is well known from fungal side chaindesaturation of sterols (25, 32) and was also suggested fornonyl chain-hydroxylated metabolites of 4-n-NP in plant cellcultures (42). The primary hydroxylation of branched alkylchains of t-NP isomers and further oxidation to aldehyde orketo compounds and carboxylic acid derivatives (Fig. 4 andTable 4) resembles the initial steps in the metabolism of n-alkylbenzenes in filamentous fungi (14) and yeast (21), thecatabolism of phenylalkanes in actinomycetes (39), and thebiodegradation of 4-n-nonylphenol by the yeast Candidaaquaetextoris (52). In these organisms, -oxidation of the linearalkyl chains of parent compounds to carboxylic acid derivativeswas implicated. As could be deduced from n-alkane and fattyacid metabolism in filamentous fungi and yeasts, monooxyge-nases of the cytochrome P450 family would be potential can-didates for the presently unknown fungal enzymes introducingoxygen functions into nonyl chains (for a recent review, seereference 11). Also, fungal desaturases can be found amongthe cytochrome P450 superfamily (5, 25).

The estrogenic potential of alkyl chain-modified metabolitesof nonylphenols has thus far not been assessed. Both the phe-nol moiety and the nature of the alkyl substituent have beenshown to affect the binding of 4-n-alkylphenols to estrogenreceptors, with the interaction increasing in strength with in-creasing length and hence hydrophobicity of the alkyl chain upto nine carbon atoms (48). The introduction of oxygen func-tions into alkyl chains of nonylphenols would lower their hy-drophobicity, and hence a decrease in the endocrine activity ofcorresponding metabolites would be expected. This has notbeen investigated thus far. Besides laccase reactions, fungalbiotransformation of t-NP into nonyl chain-modified metabo-lites catalyzed by intracellular enzymes may therefore not onlycontribute to the removal of the parent compound(s) but alsodiminish the associated endocrine effects in natural aquaticenvironments or during the application of fungal cells for thebiotechnological treatment of nonylphenol-contaminated wa-ters. This should be assessed in the future. Also, the previouslyreported shortening of alkyl chains of t-NP isomers upon fun-gal biocatalysis (23, 34) may generate metabolites, which couldbe further degraded by a variety of bacteria in addition to thosethat are capable of acting on the parent compound(s). As yet,



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two bacterial strains of the genus Sphingomonas, and one strainof Sphingobium amiense, Sphingobium xenophagum, Stenotro-phomonas sp., Pseudomonas mandelii, and P. veronii, respec-tively, have been demonstrated to aerobically degrade t-NP(11, 15, 16). P. putida MT4, P. veronii INA06, Pseudomonas sp.strain KL28, and Alcaligenes sp. have been shown to degrademedium-length alkylphenols (C3 to C7), whereas the degrada-tion of short-chain alkylphenols such as cresols and xylenolshas been described for a wide range of bacteria (11, 49). In-vestigations addressing such possible fungal-bacterial cooper-ations remain as a future task. However, the substantial riskthat more estrogenic t-NP isomers may be enriched in theparent compound mixture during intracellular isomer-specifict-NP degradation by fungi must also be addressed in the future.

ACKNOWLEDGMENTS

We acknowledge financial funding of C. Martin within the GermanResearch Foundation (DFG) graduate college 416.

We thank K. Smith (Roskilde, Denmark) for help with the Englishlanguage.

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quantiﬁcation of the inﬂuence of extracellular laccase and ... · (np112) were about 1.6- and...

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