Biodegradation capability of Absidia fusca Linnemann towards environmental pollutants

Chemosphere 52 (2003) 663–671 www.elsevier.com/locate/chemosphere

Biodegradation capability of Absidia fusca Linnemann towards environmental pollutants P. Guiraud a

a,*

, D. Villemain b, M. Kadri a, O. Bordjiba c, R. Steiman

a

Groupe pour l’Etude du Devenir des X
enobiotiques dans l’Environnement (GEDEXE), EA 2945 Environnement-Sant
e, UFR de Pharmacie de Grenoble, Universit
e Joseph Fourier, BP 138, 38243 Meylan cedex, France b Laboratoire d’Etude des Radiopharmaceutiques (LER), INSERM E00-08, UFR de M
edecine de Grenoble, Universit
e Joseph Fourier, 38706 La Tronche cedex, France c Institut des Sciences de la Nature, Universit
e d’Annaba, 23000 Annaba, Algeria Received 11 September 2002; received in revised form 23 January 2003; accepted 20 February 2003

Abstract The purpose of this work was to study the bioremediation capability of Absidia fusca Linnemann (Zygomycete) towards different classes of xenobiotics (lignin-derived compounds, chloroaromatic compounds, polycyclic aromatic hydrocarbons) the presence of which in contaminated soils, water and sediments poses a significant risk to the environment and human health. Two strains from different origins were compared. One was from an official collection and grown in non-inducing conditions, while the other was isolated during the course of the survey of fungal flora in a polluted soil from Annaba (Algeria). All data were analyzed and results validated via a statistical treatment. We showed the effect of the factors studied (origin of the strain, xenobiotic) but also the interactions between these factors. The strain of A. fusca isolated from a polluted soil was able to efficiently degrade most of the xenobiotics tested, particularly: pentachlorophenol, phenol, catechol, guaiacol and ferulic acid. This property also existed in the other strain but at a very low level. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Xenobiotics; Biodegradation; Absidia fusca; Polycyclic aromatic hydrocarbons; Phenolic compounds; Chloroaromatic compounds

1. Introduction In soil, environmental organic pollutants undergo various abiotic and biotic transformation reactions. Concerning the biotic processes, the microbial degradation of xenobiotics has been the subject of numerous studies over many years. Some fungi can be very efficient biodegradative agents but some others are very sensitive and can be used as biomarkers. The toxicity of a

* Corresponding author. Tel.: +33-4-7604-1032; fax: +33-47676-5664. E-mail address: [email protected] (P. Guiraud).

chemical toward micromycetes may lead to vegetative mycelial growth inhibition but also to side effects such as a modified pigmentation, mycelium viscosity, perturbation in sexual and/or vegetative reproduction cycles (Narayanarao et al., 1972; Guiraud et al., 1995). However, biodegradation activity is usually not inhibited in microorganisms isolated from contaminated sites (Vogel, 1996). Soil microorganisms are not equally resistant to high concentrations of chemicals (Bouwer and Zehnder, 1993; Launen et al., 1995). Some of them are very sensitive and do not grow when toxic compounds are present in high concentration or constitute a low carbon and energy source, while other are able to adapt. The latter usually exhibit specific genes encoding enzymatic systems allowing the metabolism or at least the

0045-6535/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00229-7

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transformation of contaminants (Bouwer and Zehnder, 1993). Numerous fungi can persist in a polluted milieu, either in a developing state or as dormant forms such as chlamydospores produced for example in Mucorales. The species able to resist and to grow are naturally selected in contaminated soils and often valuable for a bioremediation purpose (Chafa€ı, 1996; Bordjiba et al., 2001). The purpose of this work was to study the bioremediation capability of Absidia fusca Linnemann (Zygomycete) towards different classes of xenobiotics (lignin-derived compounds, chloroaromatic compounds, polycyclic aromatic hydrocarbons (PAHs)) the presence of which in contaminated soils, water and sediments poses a significant risk to the environment and human health. To our knowledge, nothing was reported in the literature concerning the transformation and/or degradation of xenobiotics by this species, except our recent article (Bordjiba et al., 2001). Two strains from different origins were compared. One was from an official collection and grown in ‘‘non-inducing’’ conditions, while the other was isolated during the course of the survey of fungal flora in a polluted soil from Annaba (Algeria).

2. Materials and methods

2.2. Chemicals Anthracene (AN), fluoranthene (FL), protocatechuic acid (PA), syringic acid (SA) and syringaldazine were purchased from Sigma–Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Guaiacol (G) and o-anisidine were from E. Merck AG (Darmstadt, Germany). Ferulic acid (FA), vanillic acid (VA), gallic acid and benzidine were obtained from Fluka (Buchs, Switzerland). Pentachlorophenol, sodium salt (PCPNa) and pentachloronitrobenzene (PCNB) were purchased from Janssen (Beerse, Belgium). Agar and malt extract were respectively from Coop
erative Pharmaceutique Francßaise (Melun, France) and Difal (Villefranche-sur-Sa^ one, France). Other products were from Prolabo (Paris, France). 2.3. Media and culture conditions MEA used for cultures contained: malt extract 15 g l
1 –agar 15 g l
1 . Galzy and Slonimski (1957) liquid synthetic medium (GS) was slightly modified by adding glucose at a final concentration of 5 g l
1 . The media were sterilized by autoclaving (20 min, 121 °C) before use. The strains were reactivated on MEA medium for 8 days, before each experiment. 2.4. Determination of extracellular enzymatic activities by plate assays

2.1. Fungal strain isolation A. fusca 1 (CBS 102.35, Baarn, The Netherlands) was from pine forest soil (Germany) while A. fusca 2 was isolated in our laboratory from a polluted soil from Algeria (Bordjiba et al., 2001). This contaminated soil came from a parcel treated by herbicides and fungicides, where tomato and potato were cultivated (El Krous, 30 km east from Annaba). The main pollutants found in this soil were the herbicides: metribuzin, metobromuron, linuron, and metamitron associated to the fungicides: propineb, maneozeb, and sandofan. Soil samples were taken from 1 to 10 cm depth and collected in sterile tubes. About 20 samples were taken and mixed to obtain a representative mycoflora of the parcel. Samples were stored at +4 °C until analysis. The isolation of the strains was accomplished by using the soil plates method of Warcup (Parkinson and Waid, 1960): aliquots of the soil samples were dispatched into Petri dishes (90 mm diameter) and the sterile medium MEA (malt extract 1.5%–agar 1.5%) added by chloramphenicol (0.05%) was poured over. Dishes were incubated at 22, 37, 45 and 55 °C and the fungal strains were isolated as soon as colonies appeared. For each condition five dishes were used and the whole experiment was done in triplicate. After identification, the strains were maintained in slant tubes on MEA medium. A. fusca 2 was isolated at 22 °C and was stored at 4 °C.

Production of extracellular phenoloxidases (POx ) was assayed on MEA medium as previously described (Guiraud et al., 1992a; Seigle-Murandi et al., 1992a). POx were detected by direct application of 10 reagents to the mycelium. Benzidine, guaiacol, o-anisidine, pyrogallol, a-naphtol, p-cresol, tyrosine, gallic acid were used as 0.1 M solutions in ethanol. Syringaldazine was used as a 0.1% (w/v) solution in ethanol to which a 0.03% hydrogen peroxide aqueous solution could be added to demonstrate peroxidase activity; the R56 reagent was an ethanolic solution containing 11.5% amidopyrine, 2.0% N,N-diethylaniline, 2.5% benzoic acid. Reactions were read after 20 min (syringaldazine  H2 O2 ) or 24 h (other reagents). Controls were drops of ethanol applied to mycelia and drops of reagents on fungus-free medium. These enzymatic assays were performed with 8–12 dayold cultures, grown and incubated at 24 °C. 2.5. Degradation assays To obtain sufficient inoculum for liquid cultures, strains were grown for 1–2 weeks on MEA medium. The fungi were aseptically inoculated (mycelium and spores) in GS liquid medium (glucose 5 g l
1 , pH 4.5), previously sterilized by autoclaving for 20 min at 121 °C. The 125 ml Erlenmeyer flasks containing 25 ml of inoculated medium were incubated at 22–24 °C under shaking

P. Guiraud et al. / Chemosphere 52 (2003) 663–671

(180 rpm, orbital shaker), for 2 days. At this stage, no glucose remained in the medium. Ethanol stock solutions of the phenolic compounds (2.5%) were added to 2-day-old cultures to obtain a final concentration of 0.5 g l
1 . PCPNa (0.5%) was dissolved in distilled water and PCNB (0.5%) in dimethylsulfoxide, their final concentration in the culture medium was 0.1 g l
1 . Dimethylsulfoxide stock solutions of AN and FL were sterilized by filtration through 0.2 lm Millipore membranes, and added to the final concentration of 0.01 g l
1 . The depletion of the compounds was evaluated after 4 days of cultivation at 22–24 °C. Light was 1200 lx with a photoperiod of 12 h per day. Each series of experiments was run 6 times and included cell-free flasks for abiotic degradation assessment. Biotic controls consisted of fungal cultures to which the xenobiotic was added at the time of harvesting and processed immediately. Abiotic control consisted of uninoculated flasks. The two controls served to correct the loss due to binding to the mycelium or physicochemical degradation. 2.6. Analysis of biodegradation Liquid media with mycelia, containing AN or FL were filtered off and extracted with one volume of bidistilled ethyl acetate (25 ml) by rotary shaking at 180 rpm for 30 min. The extraction was repeated twice. The organic phases were pooled, dried over anhydrous Na2 SO4 and evaporated to dryness at 40 °C under reduced pressure. The residue was dissolved in acetonitrile (1.5 ml) from which, after gently vortexing and filtration through a 0.2 lm membrane filter, an aliquot of 20 ll, was removed for HPLC analysis. HPLC was performed with a liquid chromatograph (Shimadzu) equipped with a LC 6A pump, an SIL-9A automatic injector and a RF-10AXL spectrofluorescence detector. The separation column, Supelcosil TM LC-PAH 5 lm, was 4.6 mm i.d.  150 mm (Supelco Inc., Bellefonte, PA). The mobile phase was acetonitrile:water (70:30, v:v). The flow rate was 1 ml min
1 and detection was made at 280 nm (k excitation) and 450 nm (k emission) for FL and at 250 nm (k excitation) and 450 nm (k emission) for AN. Each sample was injected three times and the mean was calculated. For the other xenobiotics, the culture medium was filtered off, the aqueous phase acidified to pH 2.0 with 6 N HCl and extracted with ethyl acetate (3 v/v). Combined extracts were dried. The residue was dissolved in methanol for HPLC. HPLC was performed using the same equipment as previously described but with a UV detector (Lambda-Max model 481). For separation of phenolic compounds, the column (model H220; Soci
et
e Francßaise de Chromato Colonne, Paris, France) was 4.6 mm i.d.  250 mm in length, packed with ODS (Hypersil, 10 lm). The mobile phase was methanol: water:acetic acid (30:70:1, v/v/v), the flow rate was 1.4

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ml min
1 , and the detection was performed at 279 nm. For separation of chloroaromatic compounds, the column was 4.0 mm i.d.  300 mm, filled with l-Bondapak C18 . The mobile phase was methanol: water (90:10, v/v) pH 5 (phosphoric acid) for PCPNa or pure methanol for PCNB. The flow rate was 1 ml min
1 and peaks were detected at 230 nm for PCPNa and at 300 nm for PCNB. Peaks were identified by comparison with the reference product. Both internal and external standardization methods were used, respectively for product identification and quantification. Adsorption of the compounds onto the mycelia was determined by shaking mycelial pellets in acetonitrile for AN or FL and in methanol for the other compounds, for 30 min at 250 rpm. After filtration, the organic phase was evaporated and the residue redissolved in a small amount of HPLC grade acetonitrile or methanol. Mycelia were oven-dried (100 °C, 1 day) and weighted. 2.7. Statistical analysis Concerning biodegradation assays, data were given as mg of degraded xenobiotic per mg of dry biomass. All results were expressed as mean  standard error. Statistical evaluations were performed using multi-factorial variance analysis in the case of multi-level qualitative factors (strain, xenobiotic). This analysis was followed by comparisons of means between groups using ‘‘a posteriori’’ tests such as Student–Fischer PLSD, Bonferroni–Dunn and Tuckey–Kramer test. Differences were considered as significant at p 6 0:05.

3. Results and discussion 3.1. Extracellular POx activities in A. fusca 1 and 2 Only a slight reaction occurred with a-naphtol for A. fusca 1 and with guaiacol for A. fusca 2. As previously reported (Rahouti et al., 1995), the less specific reagents are benzidine, gallic acid, guaiacol, pyrogallol, a-naphthol and R-56 and a slight reaction with only one of them without any response with a more specific compound detecting one of the three groups of POx (laccases, peroxidases or tyrosinases) cannot reflect the presence of these enzymes. Moreover we have previously shown that Zygomycetes (which were bad POx producers) often do not produce extracellular POx at a basal state (without induction) on a solid medium (Guiraud et al., 1992a; Seigle-Murandi et al., 1992a). 3.2. Biodegradation of xenobiotics The adsorption of the xenobiotics onto the mycelium at the end of the biodegradation experiments as well as the abiotic degradation of the compounds was found to

xenobiotics removed mg/g dry biomass

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18 16 14 12 10 8 6 4 2 0 Strain 1

Strain 2

Fig. 1. Comparison of the bioremediation capability of A. fusca strains 1 and 2 towards xenobiotics. Results are expressed as mean  standard error; value significantly different from strain 1:  p < 0:0001.

be minor. Values obtained never exceeded 5% and were taken into account in the final calculations. Controls also showed that no adsorbed xenobiotic could be extracted from the glass walls. On the whole, a significant difference was observed between the behavior of strain 1 and 2 towards xenobiotics (Fig. 1 and Table 1). Strain 2 was the most efficient at removing xenobiotics from the culture medium. The results of the bioremediation assays conducted with the two strains of A. fusca are given in Figs. 2–4. The differences between the strains and the occurrence of new peaks on the HPLC profiles together with the decrease of the peak corresponding to the xenobiotic (data not shown) were in favor of a biodegradation activity of A. fusca. Identification of the metabolites produced was not undertaken in this work. PAHs are fused ring aromatic compounds formed during the incomplete combustion of almost any organic material and ubiquitously distributed in the environment (Menzie et al., 1992; Cerniglia, 1993). Some of them are considered as dangerous substances as a function of their toxic and mutagenic or carcinogenic potentialities (Menzie et al., 1992; Nadon et al., 1995). PAHs are hydrophobic compounds, whose persistence within ecosystems is due to their low aqueous solubility (Chaudhry, 1994). Knowledge on fungal degradation of

Fig. 2. Degradation of PAHs by A. fusca strains 1 and 2. Results are expressed as mean  standard error; value significantly different from strain 1:  p < 0:05; value significantly different from anthracene: $$$ p < 0:0001.

PAHs is limited: they are oxidized to phenolic metabolites by cometabolic process (Cerniglia, 1993; Paszczynski and Crawford, 1995; Harayama, 1997). PAHs were not highly removed by A. fusca from the culture medium as compared to some other xenobiotics such as PCNB, phenol, catechol, guaiacol or FA (Fig. 2). ANOVA analysis of the data revealed a significant and strong interaction between the ‘‘strain’’ factor and the ‘‘xenobiotic’’ factor (p ¼ 0:0014). Reverse results were obtained depending on the strain. A better depletion of AN was observed with strain 2, while this strain was less efficient than strain 1 in the case of FL. This could indicate that the transforming/degrading systems of A. fusca involved are not totally similar for the two compounds. The Bonferroni–Dunn test (a ¼ 5%) showed that concerning AN, the difference observed between strain 1 and strain 2 was significant, while it was not for FL. Moreover, strain 1 was significantly more efficient in removing FL than AN (p < 0:0001). FL is the most abundant PAH in the environment, and is considered as a pollution indicator (Chaudhry, 1994). AN is another model compound for the PAHs degradation studies: its structure is found in carcinogenic PAHs such as benzo(a)pyrene and benzo(a)an-

Table 1 ANOVA table for the global xenobiotic removal by A. fusca Strain Xenobiotic Strain  Xenobiotic Residue

df

Square sum

Mean square

F value

p value

k

Power

1 10 10 110

1477.413 16177.978 2026.862 1118.153

1477.413 1617.798 202.686 10.165

145.343 159.153 19.940 –

<0.0001 <0.0001 <0.0001 –

145.343 1.592E3 199.396 –

1 1 1 –

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667

Fig. 3. Degradation of phenols and chloroaromatics by A. fusca strains 1 and 2. Results are expressed as mean  standard error; values significantly different from strain 1:  p < 0:0001; values significantly different from the other compounds: $$$ p < 0:0001.

Fig. 4. Degradation of phenolic acids by A. fusca strains 1 and 2. Results are expressed as mean  standard error; values significantly different from strain 1:  p < 0:0001; values significantly different from the other compounds: $$$ p < 0:0001.

thracene (M€ uncnerova and Augustin, 1994). A previous study showed that FL was more easily degraded by fungi than AN (Giraud et al., 2001), while other suggested similar results with the two molecules (Krivobok et al., 1998; Salicis et al., 1999). Most reports pointed out the high efficiency of the Zygomycete group, mostly Rhizopus arrhizus and the Cunninghamella genus (Cerniglia, 1982, 1984, 1993; Krivobok et al., 1998; Salicis et al., 1999), but also Absidia cylindrospora (Giraud et al., 2001). Our results show that A. fusca does not share this feature with other Zygomycetes and was able to degrade both compounds only in a limited extend even when isolated from a polluted milieu. A. fusca was more efficient towards the chloroaromatic compounds (Fig. 3). But again the ANOVA

analysis demonstrated a significant and strong interaction between the ‘‘strain’’ and the ‘‘xenobiotic’’ factor (p ¼ 0:0004) with reverse results. A better depletion of PCPNa was observed with strain 1, while this strain was less efficient than strain 2 in the case of PCNB. The Bonferroni–Dunn test (a ¼ 5%) showed that all these results were significant (p < 0:0001) confirming the conclusions of Student–Newman Keuls test (Table 2) and PLSD Fisher test (Table 3). In addition, for both strains the degradation of PCPNa was significantly better than that of PCNB (p < 0:0001). PCNB, introduced as a fungicide around 1930, is used in France mainly against the genera Botrytis, Rhizoctonia, Fusarium and Tilletia (Ingham, 1985). This compound is particularly persistent in soil, and its microbial degradation

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Table 2 Comparison of the bioremediation capability of strains 1 and 2 (A. fusca) using the Student–Newman Keuls test Compounds

Mean amount removed (mg/g dry biomass)

Comparison (a ¼ 5%)

Strain 1

Strain 2

Mean difference

Phenols Phenol Catechol Guaiacol

1.28 12.49 0.11

16.82 20.47 23.51

15.54 7.98 23.40

6.40 4.38 6.60

<0.01 <0.01 <0.01

S S S

Phenolic acids FA Protocatechuic acid Syringic acid VA

28.18 3.50 3.80 3.47

36.97 12.38 8.39 8.45

8.79 8.88 4.59 4.99

4.38 5.85 4.81 5.69

<0.01 <0.01

S S NS NS

Chloroaromatics PCPNa PCNB

41.49 4.30

33.61 11.36

7.89 7.06

4.38 4.81

<0.01 <0.01

S S

2.50 4.23

3.41 3.58

0.91 0.65

3.65 4.38

PAHs Anthracene Fluoranthene

Critical difference

Conclusion p

NS NS

Table 3 Comparison of the bioremediation capability of strain 1 and 2 (A. fusca) using the PLSD Fisher test Mean amount removed (mg/g dry biomass)

Comparison (a ¼ 5%)

Strain 1

Strain 2

Mean difference

Phenols Phenol Catechol Guaiacol

1.28 12.49 0.11

16.82 20.47 23.51

15.54 7.98 23.40

3.65 3.65 3.65

<0.0001 <0.0001 <0.0001

S S S

Phenolic acids FA Protocatechuic acid Syringic acid VA

28.18 3.50 3.80 3.47

36.97 12.38 8.39 8.45

8.79 8.88 4.59 4.99

3.65 3.65 3.65 3.65

<0.0001 <0.0001 0.0142 0.0078

S S S S

Chloroaromatics PCPNa PCNB

41.49 4.30

33.61 11.36

7.89 7.06

3.65 3.65

<0.0001 <0.0001

S S

2.50 4.23

3.41 3.58

0.91 0.65

3.65 3.65

0.6233 0.7231

NS NS

Compounds

PAHs Anthracene Fluoranthene

especially by fungi has been poorly studied. However, an extensive study was conducted on more than a thousand strains of micromycetes belonging to different taxonomic groups, followed by more detailed studies on selected species (Steiman et al., 1992; Lievremont et al., 1996; Mora Torres et al., 1996). The Mucedinaceae and the

Critical difference

Conclusion p

Zygomycetes were the most performant groups. However, the 3 species (9 strains) of Absidia tested (A. cylindrospora, A. glauca and A. spinosa) were not among the most interesting. Our results showed that A. fusca also exhibits limited interest for the degradation of this compound.

P. Guiraud et al. / Chemosphere 52 (2003) 663–671

The efficiency of A. fusca to degrade PCPNa was considerably better. The fungal degradation of this antifungal pesticide was studied in various taxonomic groups of fungi: Basidiomycetes (Mileski et al., 1988; Lamar et al., 1990), Deuteromycetes (Bollag and Loll, 1983) and more recently a wider study was conducted in different groups including Zygomycetes (Seigle-Murandi et al., 1992b, 1993). When comparing the average degradation values, the Zygomycetes, including the genus Absidia, were found the most efficient (Seigle-Murandi et al., 1992b, 1993). A comparative study of the biodegradative capability of fungi for PCP and PCNB revealed that A. cylindrospora, A. glauca and A. spinosa were all more efficient towards PCP (Seigle-Murandi et al., 1992c). Our results with A. fusca are in agreement with these observations and the discrepancy between PCPNa and PCNB degradation was higher than that reported for the other Absidia species. The best degradation activity by A. fusca was obtained with the lignin-derivative phenols and phenolic acids (Figs. 3 and 4). For the first group of phenolic compounds, an interaction between the ‘‘strain’’ and the ‘‘xenobiotic’’ factor (p < 0:0001) was revealed by the ANOVA analysis. The amount of xenobiotic removed was higher with strain 2 and this was particularly obvious for guaiacol and phenol. Strain 1 was almost unable to degrade these compounds, while it was able to degrade catechol. All the differences observed were significant except that between phenol and guaiacol with strain 1 and between catechol and guaiacol for strain 2 (Fig. 3). For the second group (Fig. 4), no significant interaction between the ‘‘strain’’ and the ‘‘xenobiotic’’ factor was noticed. The ‘‘strain’’ effect was significant (p < 0:0001): strain 2 degraded more the xenobiotics than strain 1. The ‘‘xenobiotic’’ effect was also significant (p < 0:0001): FA was considerably more easily degraded than the other compounds by both strains. Lignin-derived compounds are rejected in the effluents of numerous industries and may be found in water and sediments. Some of them, including those chosen in this study, were extensively used as models to investigate the microbial resistance and metabolism of these compounds (Eriksson et al., 1990). On solid medium, toxicity evaluation of seven phenolic compounds towards fungi belonging to different taxonomic groups has shown that the most toxic were phenol, FA, guaiacol and catechol, the least toxic being protocatechuic, vanillic and syringic acids (Guiraud et al., 1995). A. fusca, mostly strain 2, was able to efficiently degrade the most toxic of these compounds. It has been demonstrated that in white-rot fungi (Basidiomycetes), POx play a major role in the degradation of lignin and its phenolic components and most have received great attention (Aust, 1990; Eriksson et al., 1990; Barr and Aust, 1994). However, concerning

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other taxonomic groups (Ascomycetes, Deuteromycetes, Zygomycetes) the correlation between POx production and lignin models degradation has been less studied and remains controversial (Rahouti et al., 1989; Guiraud et al., 1992a,b; Seigle-Murandi et al., 1992a). As for phenolic compounds, a high correlation between ligninolytic activities (evaluated via extracellular POx activity) in cultures of white-rot fungi and the ability to biodegrade PAH exists (Field et al., 1992; Sack and G€ unther, 1993). Our previous results (Giraud et al., 2001), as well as those of Sack and G€ unther (1993) tend to indicate that this correlation does not exist for other groups of fungi. Other enzyme systems, including cytochrome P-450 monooxygenase, may be induced and responsible for degradation of aromatic compounds (Dutta et al., 1983; M€ uncnerova and Augustin, 1994). The involvement of POx in the biodegradation of PCP and chlorophenols is controversial. Phanerochaete chrysosporium was able to degrade PCP when cultivated in nutrient nitrogen-limited medium that is also necessary for the induction of its lignin-degrading system (Mileski et al., 1988). But, some degradation occurred in nutrient nitrogen-sufficient medium which means that another degrading system may also be implicated. No correlation was found between PCP degradation and extracellular POx activity by Seigle-Murandi et al. (1993) when studying a large number of fungi. As noticed for PCP, no correlation was found between POx and degradation, when fungi were investigated for their ability to degrade PCNB (Steiman et al., 1992). The degradation of PCNB depended on the taxonomic groups without regard to POx activity. The very efficient Zygomycetes did not exhibit any POx activity under the experimental conditions used. The present results confirmed these conclusions since A. fusca was able to degrade several of these xenobiotics and this property was specially obvious for the strain isolated from a polluted soil although no POx activity was detected.

4. Conclusion Fungal biotransformation and/or degradation of environmental xenobiotics has received little attention except for some groups such as Basidiomycetes and particularly white-rot fungi (Pointing, 2001). This work demonstrates that fungi from other taxonomic groups may contribute to the degradation of various pollutants and so to the remediation of contaminated soils, sediments and water. It confirms that some Zygomycetes can be particularly efficient, with a wide spectrum of action. In a previous report we noted the ability of A. fusca to degrade different classes of herbicides (the triazins: metribuzin and metamitron, and the phenylurea: metobromuron and linuron), some of them being

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very resistant to other fungi. This capability existed in strain 1, but was considerably increased in strain 2 (Bordjiba et al., 2001). The present work confirms that strain 2 is able to metabolize xenobiotics with a high efficiency. The polluted environment has probably favored the selection of a strain expressing low specific but efficient enzymatic systems, able to transform and/or degrade a large panel of molecules. Adaptation of fungi to a contaminated environment is species dependent and some strains may be selected and act together with bacteria and plants to remove xenobiotics by adsorption, biotransformation and/or biodegradation. This study may contribute to allow the improvement of decontaminating systems such as constructed wetlands for water treatment or other bioremediating ecosystems, which may be enriched in efficient fungi having biodegradative potentialities adapted to the xenobiotics present. References Aust, S.D., 1990. Degradation of environmental pollutants by Phanerochaete chrysosporium. Microb. Ecol. 20, 197–209. Barr, D.P., Aust, S.D., 1994. Mechanisms white-rot fungi use to degrade pollutants. Environ. Sci. Technol. 28, 78A–87A. Bollag, J.M., Loll, M.J., 1983. Incorporation of xenobiotics into soil humus. Experientia 39, 1221–1231. Bordjiba, O., Steiman, R., Kadri, M., Sage, L., Guiraud, P., 2001. Removal of the herbicides: metribuzin, metobromuron, linuron and metamitron from liquid media by fungi isolated from a contaminated soil. J. Environ. Qual. 30, 418–426. Bouwer, E.J., Zehnder, J.B., 1993. Bioremediation of organic compounds putting microbial metabolism to work. Bioremediation 11, 360–367. Cerniglia, C.E., 1982. Initial reactions in the oxidation of anthracene by Cunninghamella elegans. J. Gen. Microbiol. 128, 2055–2061. Cerniglia, C.E., 1984. Microbial transformation of aromatic hydrocarbons. In: Atlas, R.M. (Ed.), Petroleum Microbiology. Macmillan Publishing Company, New York, pp. 99– 128. Cerniglia, C.E., 1993. Biodegradation of polycyclic aromatic hydrocarbons. Curr. Opin. Biotechnol. 4, 331–338. Chafa€ı, D., 1996. Micromycetes des s
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