Biodegradation of micropollutant naproxen with a selected fungal strain and identification of metabolites

2.1 Chemicals

Naproxen-Na (CAS NO: 26159-34-2) and 1-aminobenzotriazole were obtained from Sigma-Aldrich.

2.2 Microorganisms

Phanerochaete chrysosporium ME446, Funalia trogii ATCC 200800, Aspergillus niger NRRL 328, and Yarrowia lipolytica NBRC1658 were used to compare removal capabilities of naproxen sodium salt. Fungal strains were kept as slopes on potato dextrose agar for up to 20 days at 4°C.

2.3 Culture medium

Two different culture mediums were used in this study. Modified Vogel’s medium was used for Funalia trogii, Aspergillus niger, and Yarrowia lipolytica, which is composed of per liter: 20 g glucose, 10 mL Modified Vogel’s medium and 100 μL trace elements solution. Modified Vogel’s medium is composed of (L⁻¹): 150 g Na₃ citrate 5·H₂O, 250 g KH₂PO₄, 100 g NH₄NO₃, 10 g MgSO₄·7H₂O, 5 g CaCl₂·2H₂O. Trace elements solution contains (100 mL): 5 g citric acid·H₂O, 5 g ZnSO₄·7H₂O, 1 g Fe(NH₄)₂(SO₄)₂·6H₂O, 0.25 g CuSO₄·5H₂O, 0.05 g MnSO₄·H₂O, 0.05 g H₃BO₃, and 0.05 g Na₂MoO₄·2H₂O [30]. Nitrogen-limited Vogel’s medium for Phanerochaete chrysosporium, contained 50 mg L⁻¹ NH₄NO₃ as a nitrogen source, all other components of culture medium were same as stated above. All culture mediums pH were adjusted to 4.7.

2.4 Removal experiments

One milliliter suspended mycelia of Funalia trogii (approximately 0.1 g, wet weight) and one milliliter spore suspension of Phanerochaete chrysosporium (1 mL; 0.5 absorbance unit at 650 nm) were inoculated into 250-mL Erlenmeyer flasks. Cultures of these fungi were precultured for 7 days at 30°C (150 rpm).

One milliliter Yarrowia lipolytica cell suspension (1 mL: 1 absorbance unit at 650 nm) and one milliliter Aspergillus niger spore suspension (1 mL; 1.2×10⁶ spore) were inoculated into 250-mL Erlenmeyer flasks and then cultures precultured for 3 days at 30°C (150 rpm). All experiments were conducted using 99-mL culture medium.

After preincubation periods, 1-mL stock solution of naproxen in methanol was added into flasks to give desired final concentration of pharmaceutical (50 mg L⁻¹). In order to determine most efficient fungal strain, all cultures incubated with 50 mg L⁻¹ naproxen on rotary shaker (30°C, 150 rpm) for 48 h.

To avoid photo transformation reactions, all the experiments were carried out in the dark. Each experiment was prepared triplicate, control groups included abiotic and heat killed organisms.

2.5 Experiments with cytochrome P450 inhibitor

1-Aminobenzotriazole (ABT) is a suicide inhibitor of cytochrome P450 and chloroperoxidases. Before addition of naproxen, 2 mM ABT was added to the culture medium, in order to reveal the role of cytochrome P450.

2.6 Isolation of metabolites

After biotransformation of naproxen, the media was extracted with the mixture of CH₂Cl₂:MeOH (93:7), evaporated and directly transferred to an open column with silica gel 60 (0.04–0.063 mm) and eluted with first only CH₂Cl₂, then the mixture of CH₂Cl₂:MeOH (100:5) with gradient elution.

2.7 LC/MS analysis

The LC/MS spectra were taken on a Waters Micromass ZQ connected with Waters Alliance HPLC, using ESI(-) method, with C-18 column. The HPLC of LC/MS was carried out on a column XTerra^®MS C-18 (4.6 mm×250 mm, 5 μm) with WATER:MeOH:% 0.1 HCOOH in acetonitrile (37:53:10) as mobile phase. The flow rate was 0.55 mL/min, the injection volume was 4 μL and the running time 20 min. The eluate was monitored by a photo-diode array detector at 254 nm. The analytical condition of mass was as follows: capillary voltage: 3.71 kV, cone voltage: 29 V, source temperature: 100°C:desolvation temperature: 350°C.

2.8 NMR analysis

For the structural elucidation of isolated metabolites, low-resolution LC/MS and NMR spectroscopy have been used. ¹H (400 MHz) and ¹³C (100 MHz) NMR spectra were recorded employing a VARIAN MERCURY AGILENT 400 MHz FT spectrometer, with CD₃OD as solvent. Chemical shifts (δ) are in ppm relative to TMS.

2.9 Mycelial dry weights

Mycelial weight was determined by filtering the content of the flasks through preweighed filter paper (Whatman) and then filter paper was left to dry at 40°C for 48 h.

3.1 Selecting most efficient strain

Aspergillus niger, Funalia trogii, Phanerochaete chrysosporium, and Yarrowia lipolytica were assessed in terms of naproxen degradation capabilities. According to LC–MS data, A. niger was found to be most efficient fungal source with 98% naproxen removal rate within 48 h (Figure 1).

Figure 1:

Naproxen removal capabilities of A. niger, P. chrysosporium, F. trogii, and Y. lipolytica (After 48 h incubation).

While living Aspergillus niger (0.74 g dry weight) cells were able to remove naproxen in high percentage (98%), heat killed Aspergillus niger cells (0.69 g dry weight) reached barely to a 19% removal rate in 48 h (Figure 2). Therefore, A. niger removes naproxen through biodegradation rather than adsorption.

Figure 2:

Naproxen removal rates for living and heat-killed Apergillus niger cells.

White-rot fungi are good candidate for biodegradation of environmental pollutants and many studies reported that these fungi are able to degrade wide variety of pollutants by means of their nonspecific ligninolytic enzymes [31]. A previous study showed that white-rot fungus Trametes versicolor almost completely degrade naproxen (10 mg L⁻¹) in 24 h [6]. In addition, another study demonstrated that Trametes versicolor remove significant amount of naproxen (0.1 mg L⁻¹) in the sterile soil environment in 24 h and the authors suggested that T. versicolor could be used for soil-bioaugmentation processes [17]. Rodarte-Morales et al. reported that 1 mg L⁻¹ naproxen was totally degraded by P. chrysosporium at day 4th [32]. Immobilized Phanerochaete chrysosporium cells almost completely degrade 1 mg L⁻¹ naproxen in 7 days [33]. Eibes et al. achieved 80% removal of naproxen using versatile peroxidase which was obtained from Bjerkandera adusta [34]. While, in our study, two white-rot fungal strains Funalia trogii and Phanerochaete chrysosporium showed lowest removal percentage in 48 h (Figure 1). Consequently, A. niger was chosen as the most efficient fungus in terms of naproxen degradation capability. A. niger is a filamentous aerobic fungus. A. niger is able to grow in the range of various pH (1.4–9.8) and temperature (6–47°C) [26]. A. niger could degrade environmental pollutants such as herbicide chlorimuron-ethyl, chlorsulfuron, and metsulfuron-methyl [28], [35]. Due to A. niger’s growth ability in a wide range of pH and temperature, we think that A. niger is a good candidate for naproxen removal applications. In addition, in our study, we showed that A. niger could remove much higher concentrations of naproxen (50 mg L⁻¹) than previous studies.

3.2 Identification of degradation products

In order to identify the degradation by-products of naproxen, LC/MS, ¹HNMR, and ¹³CNMR analyses were performed. Metabolite 1 (t_R 6.99 min), metabolite 2 (t_R 8.35 min), and untransformed naproxen (Figure 3A) were detected by LC/MS (ESI-) technique of the direct extraction of medium. Naproxen gave a molecular ion of [M–H] at m/z 229 and the adduct ion [2M–H] at 459 m/z (Figure 3B).

Figure 3:

(A) LC chromatogram relevant to naproxen after 48-h incubation with Aspergillus niger, (B) Full scan ESI (–) mass spectrum for untransformed naproxen.

Metabolite 1 (M1) (yield 14%) exhibits three major ions at m/z 215 (100%), 431 (19%), and 171 (22%), which are corresponded to molecular ion [M–H], to the adduct ion [2M–H] and ion [M-COOH], respectively (Figure 4). Major ion at m/z 215, 14 mass units lower than naproxen which was identified as O-desmethylnaproxen (C₁₃H₁₂O₃). This compound was identified as a product of fungal transformation reactions by several studies [6], [36], [37]. ¹H-NMR and ¹³C-NMR results confirmed chemical structure of M1 (Table 1).

Figure 4:

Full scan ESI (–) mass spectrum for metabolite 1 (O-desmethylnaproxen).

Table 1:

Chemical structures and ¹H-NMR and ¹³C-NMR data for fungal transformation metabolites of naproxen.

Metabolite	1H-NMR (CD3OD) 400 MHz δ ppm	13C-NMR (CD3OD) 100 MHz δ ppm
M1 (O-desmethylnaproxen)	1.51 (d, 3H, J=7.2Hz, -CH3) 3.79 (q, 1H, J=7.2Hz, -CH-) 7.02 (dd, 1H, J=8,6 & 2.4Hz, H-7) 7.06 (d, 1H, J=2.4Hz, H-5) 7.35 (dd, 1H, J=8,4 & 1.6Hz, H-3) 7.58 (d, 1H, J=8.4Hz, H-4) 7.63 (s, 1H, H-1) 7.66 (d, 1H, J=8.4Hz, H-8)	179.3, 156.6, 137.3, 135.6, 130.5, 130.1, 127.7, 127.3, 127.1, 119.6, 109.9, 47.1, 19.3
M2 (7-hydroxynaproxen)	1.48 (d, 3H, J=7.2Hz, -CH3) 3.72 (q, 1H, J=7.2Hz, -CH-) 3.88 (s, 3H, -OCH3) 6.81 (d, 1H, J=1.6Hz, H-5), 7.06 (dd, 1H, J=9,2 & 2.8Hz, H-3) 7.18 (s, 1H, H-8), 7.46 (d, 1H, J=2.8Hz, H-1), 7.62 (d, 1H, J=9.2Hz, H-4)	178.9, 158.6, 153.9, 138.1, 131.8, 130.2, 126.5, 120.1, 118.5, 109.3, 101.5, 55.8, 46.9, 19.2

Metabolite 2 (M2) (yield 84%) exhibits two major ions at m/z 245 (29%) and 201 (100%), which are corresponded to molecular ions [M–H] and [M–COOH], respectively (Figure 5). Molecular ion at m/z 245 [M–H] is 16 mass unit higher than naproxen. This ion should be formed by addition of one hydroxyl group to the aromatic moiety of naproxen molecule. Actually, the NMR data of M2 confirmed that hydroxylation has been occurred at 7 position of naproxen (Table 1). Thus, metabolite 2 was identified as 7-hydroxynaproxen (C₁₄H₁₄O₄), which was previously reported as a fungal metabolite of naproxen [37].

Figure 5:

Full scan ESI (–) mass spectrum for metabolite 2 (7-hydroxynaproxen).

3.3 Removal mechanism of naproxen

In the first part of this study, Aspergillus niger was the efficient strain in terms of naproxen removal potential and two fungal transformation products of naproxen, O-desmethylnaproxen and 7-hydroxynaproxen were identified using LC/MS and NMR analyses.

First of all, we had to demonstrate localization of enzymes/enzyme which were responsible of transformation reactions. In order to show whether extracellular enzymes responsible of formation of metabolites, culture supernatant was used for extracellular enzyme source.

We incubated naproxen with A. nigerculture supernatant for 48 h, and we could not determine formation of any metabolites. These results indicate that intracellular enzymes or enzyme have a role biotransformation of naproxen. Cytochrome P450 (CYP) isoforms CYP2C9 and CYP1A2 are responsible for O-demethylation of naproxen in the human liver [38]. Fungal cytochrome 450 catalyzes many transformation reactions including hydroxylation, epoxidation, and O-demethylation. CYP53A1 of A. niger catalyze O-demethylation of 3-methoxybenzoate derivative [39]. Moreover, previous studies showed that fungal cytochrome P450 enzyme system have an important role on transformation of naproxen [36], [37]. To ascertain the role of cytochrome P450, CYP450 inhibitor 2 mM 1-aminobenzotriazole was added in culture medium before addition of naproxen.

After 48 h incubation, we observed that 1-aminobenzotriazole completely inhibited formation of O-desmethylnaproxen and 7-hydroxynaproxen. A previous study reported that Cunninghamella species transformed naproxen into desmethylnaproxen and desmethylnaproxen-6-O-sulfate via mammalian analog cytochrome P450 enzyme system [36]. Cytochrome enzymes are usually involved in various hydroxylation reactions. Aspergillus nidulans can catalyze hydroxylation of phenylacetate by means of cytochrome P450 monooxygenase [40]. Thus, we suggest that A. niger can perform biotransformation of naproxen by CYP450 enzyme system (Figure 6).

Figure 6:

Proposed pathway of naproxen transformation.