White-Rot Fungus Implications in Clofibric Acid Biodegradation

WHITE-ROT FUNGUS IMPLICATIONS IN CLOFIBRIC ACID BIODEGRADATION

Received for publication, February 23, 2014 Accepted, April 23, 2015

CLAUDIA POPA (UNGUREANU) 1,2, TIBERIUS BALAES1, LIDIA FAVIER3, CĂTĂLIN TĂNASE1, GABRIELA BAHRIM2 1Alexandru Ioan Cuza University, Faculty of Biology, Bd.Carol I, No. 20 A, 700505 Iași, România 2Dunărea de Jos University, Faculty of Food Science and Engineering, 47 Domnească St., 800008 Galați, România 3Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Université Européenne de Bretagne, General Leclerc Av., CS 50837, 35708 Rennes Cedex 7, France Corresponding author. Mailing address: Dunărea de Jos University, Domnească Street, no 111, 800821, Galați, Romania; Tel.: +40 336.130.184.; E-mail: [email protected]

Abstract The clofibric acid (CLF) is the active compound of blood lipid regulators, which act by lowering serum triglyceride production. This xenobiotic compound is highly persistent in the aquatic environment and passes unchanged or poorly transformed in wastewater treatment plants. There was demonstrated the implications of filamentous fungi in biodegradation of organic compounds with aromatic chemical structure because they are able to produce extracellular phenol oxidases. This work performed a screening of four white-rot fungi, Trametes versicolor, Trametes pubescens, Lenzites betulina and Irpex lacteus based on their potential for extracellular laccase biosynthesis and biodegradation of 15 mg L-1 of CLF, after 14 days of incubation in submerged aerobe conditions by cultivation on a minimal liquid medium containing pharmaceutically active compound. The obtained results reported the ability of the tested fungal strains to remove CLF in percentages varying from 32% to 54%. Further researches target the optimization of the biotechnological conditions for CLF biodegradation in order to enhance the bioremediation yield especially in poluated aquatic system.

Keywords: White-rot fungi (WRF); Clofibric acid (CLF); Biodegradation

1. Introduction Many pharmaceutically active compounds (PhACs) have emerged as main water pollutants, based on their toxicity of biological systems, even in concentrations of ng L-1 up to µg L-1. It is important to highlight the potential impact of the release of pharmaceuticals into the environment, since these compounds are designed to affect biochemical and physiological functions on aquatic organisms. Among PhACs, clofibric acid (2-(4-Chlorophenoxy)-2- methylpropanoic acid) (CLF) is the main pharmacologically active metabolite in pharmaceutical products used for controlling blood lipid content. This drug is a recalcitrant compound and presents persistence especially in the aquatic environments. Conventional methods such as advanced oxidation process, activated carbon adsorption, and UV/H2O2 induced photolytic degradation, give better results of CLF degradation (>90%) but the main limitation is the formation of hazardous byproducts (I. Negron & Arce [1]).

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Since conventional wastewater treatment processes (WWTPs) were demonstrated to be inefficient for the degradation of recalcitrant pharmaceuticals, the implementation of advanced bioremediation techniques using biomass (microorganisms and/or plants) to degrade toxic compounds are considered as relevant because it is environmental friendly and implies relatively low costs (M. McGuiness & al. [2]). White-rot fungus (WRF) possesses a high capability to degrade a wide variety of xenobiotic compounds, such as polycyclic aromatic hydrocarbons or synthetic dyes due to their potential to produce many phenoloxidases, manganese peroxidase (MnP), lignin peroxidase (LiP), versatile peroxidase (VP) and laccase (N. Duran & E. Esposito [3]). However, the biodegradation of CLF and its derivate compounds by relevant pure cultures of microorganisms (such as fungi and bacteria) has not been widely investigated yet. Only few studies showed the ability of white-rot fungi like as Trametes versicolor, Irpex lacteus, Ganoderma lucidum and Phanerochaete chrysosporium, to degrade carbamazepine, ibuprofen and CLF, in concentration of 10 mg L-1. There are demonstrated the ability of studied strains to integrally transformation of ibuprofen while carbamazepine and clofibric acid were much more recalcitrant. Also, Trametes versicolor was the only fungus able to degrade CLF, in a high percentage close to 97% (E. Marco-Urrea & al. [4]). The aims of this work were to study the ability of some white-rot fungus strains, Trametes versicolor, Trametes pubescens, Lenzites betulina and Irpex lacteus, to biotransform CLF, in order to establish the cells resistance to PhAC toxicity and evaluation of the rate of biotransformation potential during submerged cultivation in controlled biotechnological conditions in a minimal medium composition.

2. Materials and methods 2.1. Chemicals and fungal strains Analytical grade chemical reagents, clofibric acid (CLF), methanol (HPLC grade) and ingredients for culture media formulation were purchased from Sigma-Aldrich (St. Louis, MO, USA). The white-rot fungal strains used in this study, Trametes versicolor, Trametes pubescens, Lenzites betulina and Irpex lacteus, were provided from the Cultures Collection of the Faculty of Biology, Alexandru Ioan Cuza University of Iasi. All fungi were maintained by subculturing on 2% malt extract agar slants (pH 4.5) at 4°C. Subcultures were routinely made every 30 days. For the start inoculum obtaining, 1 cm2 of mycelium growth on MEG agar slant in Petri dishes, containing (g L-1): malt extract 20, glucose 5, agar 15, was cut from colony in active growth stage as described by Levin [5].

2.2. Fungal resistance against clofibric acid toxicity Qualitative screening assays regarding the testing of fungal resistance against CLF toxicity were carried out by stationary cultivation on MEG agar medium supplemented with different concentrations of CLF (10, 15, 20 mg L-1). The plates were incubated at 25°C for 8 days and the hyphal extension of each fungus was measured daily. Hyphae extension was evaluated from the center of the colony to the edge of the plate, considering that the maximal growth corresponded to a hyphal extension of 4 cm (L. Soo-Min & al. [6]).

2.3. Clofibric acid biodegradation The CLF biotransformation of tested fungus was studied by the cultivation in submerged aerobe conditions in liquid basal medium (MM), containing (g L-1): malt extract, 15, yeast

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CLAUDIA POPA (UNGUREANU), TIBERIUS BALAES, LIDIA FAVIER, CĂTĂLIN TĂNASE, GABRIELA BAHRIM extract 5, glucose 5, MgSO4 ·7 H2O 0.5, (NH4)2 SO4 0.5, pH 5.5. This medium was supplemented with various concentrations of CLF, 10 and 15 mg L-1, respectively. A vegetative mycelial suspension of white-rot fungi was obtained by inoculation of three plugs (6 mm in diameter) of agar plugs, from the growing zone of fungi on plates, in 150 mL of MEG broth which was cultivated on a rotary shaker SI-300R Incubator Shaker (Jeio Tech, Korea) at 135 rpm, 250C for 4-5 days. Then the dense mycelial mass was aseptically blended with a homogenizer (Waring blender, Germany), to obtain homogeny mycelial inoculum. Pellets were obtaining by inoculating of 1 mL of the mycelial suspension to 150 mL of MM medium. The cultivation took places on a rotary shaker SI-300R Incubator Shaker (Jeio Tech, Korea) at 135 rpm for 5 days and 250C and then the mycelial pellets obtained were washed with deionized water. Each flask containing 150 mL of sterile MM medium was then inoculated with a 2 g of wet pellet of pure cultures (equivalent to 5.0 g L-1 dry weight biomass). Stock solution of CLF in methanol (100 mg L-1) was then aseptically added to the medium to give the desired final concentration (10 and 15 mg L-1) prior to inoculation. The cultivation took places for 14 days on orbital shaker SI-300R Incubator Shaker (Jeio Tech, Korea) at 135 rpm and temperature of 25ºC. Cultures on MM broth without added CLF and non-inoculated medium were used as biotic and abiotic controls, respectively. In all biodegradation experiments, the flasks were covered with aluminum foil to prevent photodegradation of the target compound. After incubation, culture samples were centrifuged at 10000g for 10 minutes. Supernatants were filtered through a 0.2 µm polypropylene filters (PALL Life Science, New York, USA) and then transferred to HPLC vials for subsequent high performance liquid chromatography (HPLC) analysis. In all biodegradation experiments, the samples were taken at different time intervals and evaluated for biomass yield (expressed as dry weight), CLF concentration and laccase activity as described in the following part.

2.4. Clofibric acid biosorption assay The control samples were performed to monitoring the abiotic degradation of the target molecule and also to evaluation the possible adsorption of the drug on the cell biomass. Biosorption assays were carried out using killed biomass by thermal and chemical treatment. During the thermal treatment the biomass was suspended in Erlenmeyer flasks containing liquid basal medium (MM) after which a sterilization treatment was applied at a temperature of 121oC for 15 min. Besides, the biomass thermal inactivation, the biomass of fungal cultures was chemically destroyed by the treatment with sodium azide, in a concentration of 0.2 g L-1. All experiments were performed under identical conditions to those of the biodegradation step.

2.5. Analytical procedures Mycelium dry weight Mycelium dry weight was determined by vacuum filtering the cultures through preweighed glass filters (Whatman GF/C, Maidstone, England). The filters containing the mycelial mass were placed in glass dishes and dried at 100 0C (Drying Oven Sanyo, Japan) to constant weight.

Laccase assay The culture broth was centrifuged at 1000 rpm for 10 minutes at 4oC and the cell-free supernatant was used as crude enzyme extract. Laccase (E.C.1.10.3.2) activity was determined

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in a reaction mixture of 2 mL containing 10% ABTS (2,2’-azino-bis(3-ethylthiazoline-6- sulfonate) in 0.1 M acetate buffer (pH=5.0) and measured increase in OD at wavelength of 420 nm ( 420 =3.6 x 104 M-1 cm-1) after 5 min and 25º C. One unit of enzyme activity was defined as the amount of enzyme oxidizing 1µmol of ABTS per minute (R. Bourbonnais & al. [7], C. Popa & al. [8]).

HPLC analysis Residual CLF content in cell free supernatants was performed using an HPLC Agilent 1200 Series (Santa Clara, Ca, SUA) equipped with a photodiode array (PDA) detector at wavelength of 230 nm. The separation took place by a reverse phase BDS Hypersil C18 column (150 mm x 4.6 mm, particle 5 µm). The column temperature was set at 40 0C. The CLF was eluted at a flow rate of 1 mL min-1 using isocratic conditions of 70/30 methanol/ ultrapure water acidified with 0.1% (v/v) acetic acid. The retention time was 2.4 minutes and the instrumental quantification limit (LOQ) for CLF was < 0.2 mg L-1. CLF concentration (expressed as mg L-1) was calculated using calibration curve, which was carried out by using standard solution with known CLF concentration (in the range of 10- 30 mg L-1). Biotransformation yield of CLF was calculated as follows:

C Cf 100 Biotransformation yield (%) = o Co -1 where: Co and Cf are the initial and after 14 days of fungi cultivation of CLF concentration, (mg L ).

2.6. Statistical analysis All experiments were duplicated and the data presented represented the mean values of these replicates. Data related to the CLF biotransformation was subjected to analysis of variance (one way ANOVA), using SPSS (version 19) statistical software. The differences with p < 0.05 were considered significant.

3. Results and discussion Toxicity of clofibric acid upon fungal metabolic activity Fungi belong to a very diverse group of eukaryotic organisms that populate various habitats and due to an extraordinary plasticity they can colonize different substrates using resources that are inaccessible or hardly accessible to other species, like keratin, collagen, elastin, lignin, cellulose, hemicellulose etc (S.T. Chang & P. Miles, [9]). The vegetative body of the fungi is called mycelium and it consists of septate and aseptate filamentous hyphae, which have different arrangements depending on the taxonomical position of the considered species (C. Tănase & E. Sesan, [10]). The selected strains were identified by macroscopic and microscopic characteristics using literature data from different authors (J. Erkisson & al. [11], [12], L. Hansen & H. Knudsen, [13], [14], A. Bernicchia, [15], C. Tănase & al. [16]). Some details of the main morpho-cultural and physiological characteristics of the tested fungal strains are given in Table 1.

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Table1. The morphological characteristics of the fungal strains

Classification Source of Cultural morphological aspects isolation Aerial mycelium by stationary Mycelium by submerged cultivation cultivation Irpex lacteus Primordia Generative hyphae and fibber Generative hyphae, hyaline, with hyphae, 1.5 to 2 μm rare septa, 4.5 to 6 μm diameter, diameter, with clamp connections. hyaline. Lenzites betulina Primordia Generative hyphae and skeletal Generative hyphae branched, hyphae very numerous, hyaline, septate, with clamp connections, thick refringenţ walls hyaline, of 2.2 to 4.5 μm in occasionally diameter. branched, of 1.5-3 μm diameter. Trametes pubescens Fruit bodies Generative hyphae, thick, Generative hyphae, hyaline, with strongly branched and twisted, septa, 1.5 to 4.5 μm in diameter. with clamp connection, from 3.5 to 6 μm in diameter and skeletal hyphae, thin, long, with few and simple septa, of 1.8 to 2, 2 μm thick. Trametes versicolor Primordia Generative hyphae, long, hyaline; Generative hyphae, hyaline, with skeletal hyphae, thin, hyaline, septa and clamp connections, 3.0 long, unbranched, without septa, to 4.5 μm diameter. from 1.5 to 4 μm thickness and fibber hyphae, branched, without septa.

Due the enzymatic potential of the white rot fungi to degrade a wide substrate range they can be therefore considered as potential candidates to transform CLF. The resistance at CLF toxicity of studied fungal strains was evaluated by comparing the colonial growth rate whit the control that did not contain the target molecule. However, as the CLF concentration in cultivation medium increased, the mycelium growth rate decreased to approximately 1 cm/day. Comparing with bacteria, it was found that the all analyzed fungal strains were resistant to a high (10-20 mg L-1) CLF concentration (Popa & al. [17]). Also, it was observed that no significant differences between the control and experimental plates at CLF concentration below 10 mg L-1 in case of the strains Trametes versicolor and Trametes pubescens (Figure 1a, b). The Trametes versicolor strain was extremely resistant at all tested concentrations (10, 15, 20 mg L-1) which showed higher extension, whereas strain Trametes pubescens reached the maximum hyphal extension after 6 days at concentration of 15 and 20 mg L-1, respectively. For strains Irpex lacteus and Lenzites betulina the mycelium growth was gradually inhibited at all tested concentrations (Figure 1c, d). Generally, it was observed that the target molecule did not affect the viability of fungal cells. According to the obtained results the strains Trametes versicolor, Trametes pubescens and Lenzites betulina strains were selected to be resistant at CLF toxicity and these had been used for CLF biotransformation by the cultivation in submerged cultivation as bioremediation agents.

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Control 10mg/L 4

1 Mycelium growth(cm)

0 02468 Incubation time (day) a) b)

Control 10mg/L 15 mg/L 20 mg/L Control 10mg/L 4 4 15 mg/L 20 mg/L

3 3

2 2

Mycelium growth (cm) 1 1 Mycelium growth (cm) growth Mycelium

0 0 02468 Incubation time (day) 02468Incubation time (day) c) d)

Figure 1. Inhibition of growth of the Trametes versicolor (a), Trametes pubescens (b), Irpex lacteus (c), Lenzites betulina (d) by stationary cultivation in slant agar medium with 10, 15, 20 mg L-1 CLF

Clofibric acid biodegradation by white rot selected fungi Our results revealed that less than 10 % of CLF was removed due to biosorption of the active biomass. Also, the thermal and chemical inactivated biomass of fungal cultures did not bind the target compound solubilized in the liquid basal medium in a concentration of 10 and15 mg L-1, respectively. To evaluate the impact of the initial CLF amount on the biodegradation potential of the selected strains, Trametes versicolor, Trametes pubescens and Lenzites betulina, these strains were cultivated in MM liquid basal medium supplemented different of CLF concentrations (10 and 15 mg L-1). A significant influence on the CLF biodegradation could be its concentration as pollutant in the contaminated media. The effect of initial CLF concentrations on mycelial dry weight was slight. Cultures with 15 mg L-1 of added CLF showed < 10% drop in mycelial dry weights as compared to cultures with 10 mg L-1 of added CLF. The impact of CLF toxicity was also clear from the

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CLAUDIA POPA (UNGUREANU), TIBERIUS BALAES, LIDIA FAVIER, CĂTĂLIN TĂNASE, GABRIELA BAHRIM examination of Figure 2 owing to the decrease in biomass concentration with the increase in the initial CLF amount. It was remarkable the fact that Trametes pubescens strain degrades in high percentage the drug, even though dry weight of biomass was less than observed with the other tested strains, like Trametes versicolor strain (Figure 2).

Trametes versicolor Trametes pubescens 8 6 Figure 2. Residual concentration of 4 CLF after biotransformation by the fungal cultures cultivated in aerobe 2 submerged conditions in the liquid

weight L-1) weight basal medium, (after 14 days at 25ºC 0 and 135 rpm). Error bars represent 10 mg/L 15 mg/L standard deviation of the duplicates Biomassmycelial (mg dry Clofibric acid concentration

Also, further experiments should be carried out to identify the degradation metabolites and their toxicity. After 14 days of submerged cultivation CLF from liquid medium was degraded up to 90 % by the Trametes versicolor and Trametes pubescens strains, at initial pollutant concentration of 10 mg L-1, whereas for Lenzites betulina strain the degradation yield show low levels, up to 10 %. Also, Marco-Urrea [4] reported that fungal strain of Trametes versicolor was able to degrade the CLF in a high percentage close to 97 % after 7 days of treatment in liquid medium at initial pharmaceutical concentration in medium of 10 mg L-1. In addition, a higher removal rate was achieved (80% after 6 h) by inducing hydroxyl radical production via quinoine redox cycling in this fungus (E. Marco-Urrea & al. [18]). The CLF degradation yield at an initial concentration of 15 mg L-1 was much lower. The extent of CLF degradation by Trametes pubescens strain (54%) was slightly greater than that show by Trametes versicolor strain (32%), whereas CLF was still present in the cell free culture supernatant at the end of cultivation of Lenzites betulina strain, as is showed in Figure 3.

Trametes versicolor Trametes pubescens

100 Figure 3. Residual concentration of CLF after biotransformation by the 80 fungal cultures cultivated in aerobe 60 submerged conditions in the liquid basal medium (after 14 days at 25ºC 40

and 135 rpm). Error bars represent rezidual % CLF standard deviation of the duplicates 20 0 10 mg/L 15 mg/L

Clofibric acid concentration

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Evengelista [19] tested the ability of bacteria, Pseudomonas putida, Aspergillus niger, Bacillus subtilis, Pseudomonas flourescens, Sphingomonas herbicidovorans and Rhodococcus rhodochrous to degrade CLF. It was shown that none of this microorganism was able to degrade this recalcitrant pharmaceutical compound, except the Sphingomonas herbicidovorans ATCC 700291 who converted the CLF in ethyl clofibrate. Also, Popa [20] reported that strain Streptomyces MIUG 4.89 was able to biotransform this target molecule in percentage of 14%. A considerable effect of the initial pollutant concentration was observed on the CLF removal, it decreased concomitantly with increasing initial concentration of the pollutant (C.Popa & al. [17]). There is evidence that the biodegradation rates are concentration dependent. White rot-fungus is a group of microorganisms capable of aerobically depolymerizing and mineralizing the highly resistant polyphenolic natural polymer lignin. These microorganisms produce intracellular and extracellular enzymes, which typically have low specificity for the substrate and as a result are also capable of degrading a wide variety of xenobiotic compounds ( Y. Zhang & S. Geiben [21]), such as PAHs, azo dyes and many others. Previous studies showed, the oxidation of others environmental organic pollutants, such as nonylphenol and bisphenol A by laccases from white-rot basidiomycetes (T. Fukuda & al. [22], Y. Tsutsumi & al. [23], H. Uchida & al. [24]). Thus, the role of laccase in CLF biodegradation cannot be underestimated. The production of extracellular laccase by studied fungi was also studied in correlation with biodegradation of CLF yield. As shown in Figure 4 and 5 after 14 days of cultivation in MM basal liquid medium supplemented with 10 mg L -1 CLF, extracellular laccase activity did not exceed 450 UA L-1. A positive correlation between CLF biodegradation and laccase activity was observed after long time of cultivation (i.e. 10 days). A long lag period for enzyme biosynthesis is observed for selected strains although CLF biodegradation is much faster (after 2 days).

100 500

80 400 Laccase activity activity Laccase Figure 4. CLF biotransformation yield and

60 300 L (UA extracellular laccase production by Trametes versicolor by submerged aerobe 40 200

yield (%) cultivation in MM basal liquid medium -1 -1 20 100 ) supplemented with 10 mg L CLF, after 14 days at 25ºC and 135 rpm. () laccase CLF biotransformation biotransformation CLF 0 0 activity; (♦) CLF biotranformation yield 0 2 4 6 8 10111214 Time (days)

100 500 Figure 5. CLF biotransformation 400 yield and extracellular laccase 80 Laccase activity production by Trametes pubescens 300 60 L (UA by submerged aerobe cultivation in 40 200

MM basal liquid medium -1

-1 ) supplemented with 10 mg L CLF, 20 100 after 14 days at 25ºC and 135 rpm. yield (%) () laccase activity; (♦) CLF 0 0

biotranformation yield CLF biotransformation 0 2 4 6 8 10 11 12 14 Time (days)

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After four days of cultivation a small decrease of CLF removal was observed without correlation between extracellular laccase production and degradation of CLF. Although, previous researches demonstrated that this enzyme was not involved in the degradation of CLF (N.H. Tran & al. [25]). Also, the high concentration of CLF, 15 mg L-1, has a negative influence on production of enzymes implied in biodegradation. Marco-Urrea [4] reported that intracellular enzymatic system plays a major role in the first step of CLF oxidation by Trametes versicolor strain. In addition, other ligninolytic enzymes have been assumed to be involved in pharmaceuticals compound degradation by white-rot fungi. Zhang& Geiben [21] reported a CBZ removal in percentage of 10 % after the addition of crude lignin peroxidase from P. chrysosporium, while Trametes versicolor was not able to produce the lignin peroxidase enzyme. Another enzymatic mechanism involved in degradation of pharmaceuticals compound is those involving the CYT P 450 system (cytochrome P450). Other studies showed that enzymatic system (cytochrome P-450) is involved in the degradation of biphenyl, phenol, benzo(a)pyrene, aniline and pyridine (T.J. Shrestha & al. [26]). Supplementary tests are needed in the future in order to confirm the involvement of the ligninolytic enzymes and the CYT P450 system in the oxidation of the target molecules. Larsson [27] reported that effluents from health care industry can contain high concentrations of pharmaceuticals. It is important to remark that the concentrations tested (10 and 15 mg L-1) are considerably higher than those presented in wastewater effluents (commonly in the µg L-1 range). In this study Trametes versicolor and Trametes pubescens strains showed remarkable ability to CLF biotransformation, in concentrations of 15 mg L-1. Also, the selected strains showed the highest growth potential reaching the highest biomass concentration at all of the tested CLF. These two strains have been selected for further studies to determine the suitable cultivation conditions (concentration of inoculum, carbon and nitrogen sources, incubation time, temperature, agitation rate) for CLF biotransformation, biotechnological processes optimisation and enhance the biodegradation yield. Also, the ability of a microorganism to degrade recalcitrant compounds is strongly influenced by many experimental factors such as nutritional requirements and physico-chemical cultivation conditions.

4. Conclusions This study reporting the successful possibility to use of white-rot fungus selected strains for clofibric acid removal in simple cultivation controlled conditions, similarly with wastewater treatment systems. Four ligninolytic fungal strains, Trametes versicolar, Trametes pubescens, Irpex lacteus and Lenzites betulina, were screened based their ability to grow in the presence of 10, 15, 20 mg L-1 of CLF as target molecule in wastewater bioremediation. Two strains of Trametes versicolar and Trametes pubescens were able to biotransform the pharmaceutical compound in percentage varying from 32 to 54%, respectively, during cultivation in a minimal basal medium, in aerobe conditions in submerged system in presence of 15 mg L-1 CLF. These research approaches would be useful for the practical applications of white-rot fungi in wastewater treatment for xenobiotic recalcitrant compounds removal, like pharmaceutical pollutants. Moreover, it will be also important to evaluation the adaptation of the selected strains to natural conditions in competition with wild microbiota from the activated sludge.

Acknowledgments This work was supported by the strategic grant POSDRU/159/1.5/S/133391, Project “Doctoral and Post-doctoral programs of excellence for highly qualified human resources 10396 Romanian Biotechnological Letters, Vol. 20, No. 3, 2015

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training for research in the field of Life sciences, Environment and Earth Science” cofinanced by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007 – 2013”.

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