Accepted Manuscript Biodegradation of nonylphenol by a novel enthomopathogenic Metarhizium robertsii strain Sylwia Różalska, Adrian Soboń, Julia Pawłowska, Marta Wrzosek, Jerzy Długoński PII: DOI: Reference:

S0960-8524(15)00675-6 http://dx.doi.org/10.1016/j.biortech.2015.05.011 BITE 14982

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

25 March 2015 6 May 2015 7 May 2015

Please cite this article as: Ró żalska, S., Soboń, A., Pawłowska, J., Wrzosek, M., Długoński, J., Biodegradation of nonylphenol by a novel enthomopathogenic Metarhizium robertsii strain, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.05.011

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1

Biodegradation of nonylphenol by a novel enthomopathogenic Metarhizium

2

robertsii strain

3

Sylwia Różalska1, Adrian Soboń1, Julia Pawłowska2, Marta Wrzosek2, Jerzy

4

Długoński1*

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7

1

Department of Industrial Microbiology and Biotechnology, Faculty of Biology and

Environmental Protection, University of Łódź, Poland 2

Department of Systematics and Plant Geography, Faculty of Biology, University of

8

Warsaw, Warsaw, Poland

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* Corresponding author Jerzy Długoński, tel: +48 42 635 44 65, fax: +48 42 665 58

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18, e-mail: [email protected]

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11

Abstract

12

The biodegradation of nonylphenol (NP) by a newly isolated form of the larva fungal

13

strain Metarhizium robertsii IM 6519 was investigated in this study. This isolate was

14

capable of degrading 4-n-NP, and multiple metabolites were detected. The

15

coexistence of parallel degradation pathways with versatile hydroxylation in different

16

positions of the alkyl chain is a unique feature of this strain. Moreover, several

17

metabolites previously described only in higher eukaryotes were detected in the

18

fungal cultures. The degradation process led to the mineralization of 4-n-NP (with an

19

efficiency of 36%), a great advantage of this strain that results in complete removal of

20

toxic substrate from the environment.

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Keywords: nonylphenol, Metrahizium robertsii, biodegradation, degradation

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pathway, hydroxylation

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1. Introduction

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Nonylphenols (NP) are toxic, organic pollutants classified as endocrine disrupting

26

compounds (EDCs) capable of interfering with the hormonal system of numerous

27

organisms (Brown et al. 2009; Corvini et al. 2006; Soares et al. 2008). The main

28

source of NP in the environment is the degradation of nonylphenol polyethoxylates,

29

which are one of the most widely used surfactants (Stasinakis et al. 2008; Tuan et al.

30

2013). NP has a low solubility and a high hydrophobicity, and it easily accumulates in

31

contaminated areas. The main impact of NP in the environment includes animal

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feminization at low concentrations, but its toxicity toward different organisms has

33

induced apoptosis and oxidative stress (Deng et al. 2010;Wu et al. 2009).

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Conventional or advanced wastewater treatment methods are inefficient at removing

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NP, causing a constant release of this pollutant into the environment where it is

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commonly detected in rivers, surface waters, estuaries, sediments, drinking water

37

and air (Soares et al. 2008).

38

In recent years, NP elimination from the environment by the biodegradation

39

processes has been considered as the most important mechanism. Several studies

40

have indicated the significance of microbes and microbial activity in NP utilization

41

(Brown et al. 2009; Chang et al. 2007; Corvini et al. 2006; Ojeda et al. 2013).

42

However, the data concerning the involvement of certain microorganisms, especially

43

filamentous fungi, in NP elimination are still limited. Besides, the microbial

44

degradation pathways, indicating the complete degradation process, have been

45

described only for several species (Dubroca et al. 2005; Krupiński et al. 2013;

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Różalska et al. 2010; Vallini et al. 2001).

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47

In our former paper, the morphological response to NP of the filamentous fungus

48

Metarhizium robertsii was inspected (Różalska et al. 2014). Herein, the ability of NP

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elimination by a new Metarhizium isolate was investigated. In laboratory conditions,

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this study allowed identifying metabolites and describing the 4-n-NP degradation

51

pathway.

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2. Materials and Methods

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2.1. Fungal strain, media and cultivation techniques

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The fungal strain (IM 6519) used in this work was isolated from dead larva body of

55

Agriotes sputator L. (the click beetle; found near Łódź, Poland) in accordance with

56

standard microbial protocols. After isolation, the strain was cultivated and maintained

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on ZT agar slants (Różalska et al. 2013;Słaba et al. 2013a). The images were

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acquired using an AxioCam HRc camera with an Axiovert 200 M (Zeiss, Germany)

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inverted microscope equipped with a Plan Apochromat objective (63x/1.4 oil). For

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morphological assessments, at least 200 spores and 50 phialides were automatically

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measured using AxioVision software (Zeiss, Germany).

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2.2. DNA extraction, PCR amplification, sequencing and phylogenetic analysis

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Genomic DNA was extracted, amplified and sequenced as previously described

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(Różalska et al. 2013). The full ITS region was amplified using the primer pair ITS1f

65

and ITS4 and the TEF gene with the primers EF1-728f and EF1-986r. The following

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primers were used to amplify intergenic regions: MzIGS3_1F, MzIGS3_4R,

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MzIGS2_2F, MzIGS2_3R, MzFG543igs_1F and MzFG543igs_4R (Kepler and

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Rehner 2013). Sequence data generated for this study are available from GenBank

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under the accession numbers specified in the text. The ITS, TEF, MzIGS2, MzIGS3

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and MzFG546igs sequences generated in this study were assembled with the 4

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sequences of 9 strains retrieved from GenBank and aligned in the SeaView program,

72

version 4.3.5, using the muscle method. A maximum likelihood (ML) analysis was

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performed using PHYPLIP v3.52 and PhyML v3.0 as implemented in SeaView to

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confirm the proper species identification.

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2.3. Biodegradation experiments

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Spores from 14-d-old cultures on ZT slants were used to inoculate 20 mL of mineral

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medium in 100-mL Erlenmeyer flasks, as described earlier (Różalska et al. 2010;

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Słaba et al. 2013b). The medium consisted of (g L-1): K2HPO4 (4.36), KH2PO4 (1.7),

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NH4Cl (2.1), MgSO4 × 7H2O (0.2), MnSO4 (0.05), FeSO4 × 7H2O (0.01), CaCl2 ×

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2H2O (0.03), glucose (20) and distilled water (up to 1000 mL). After 24 h of

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incubation, 2 mL of the homogenous preculture was introduced into 18 mL of a

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mineral medium, supplemented with 50 mg L−1 of 4-n-NP dissolved in ethanol (stock

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solution 20 mg mL−1). Abiotic controls (uninoculated cultures) were also prepared.

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Samples were incubated on a rotary shaker (150 rpm) at 28 °C. At 6-h intervals, the

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culture samples were withdrawn for analysis.

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2.4. Mineralization of labeled 4-n-NP

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For radioactive tests, 1 µCi of the 4-n-NP [ring-U-14C] and non-labeled 4-n-NP were

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applied to provide the final concentration of 40 mg L-1. Radioactivity was measured in

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a 1450 MicroBeta Liquid Scintillation Counter from Wallace LKB (Turku, Finland)

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using Ultima Gold and CarboSorb E scintillation solutions (PerkinElmer, USA). The

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detailed procedure of sample preparations and radioactivity calculations were

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performed according to the method described in our previous paper (Różalska et al.

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2010)

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2.5. 4-n-NP determination

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The samples were homogenized and extracted with methylene chloride and ethyl

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acetate; they were prepared for GC-MS analyses as previously described (Różalska

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et al. 2010). The analyses of the extracts were completed on an Agilent Technologies

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7890 A series GC equipped with a mass selective detector 5975 C, using the HP-

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5MS capillary column (30 m × 250 µm × 0.25 µm). The injection volume was 2 µL.

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The inlet was set to a split mode with a split ratio of 10:1 (the split flow was 10 mL

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min−1), and the temperature was maintained at 275 °C. Helium was used as a carrier

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gas. The column temperature parameters were as follows: 60 °C maintained for 2

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min, 20 °C min−1 to 300 °C and maintained for 8 min. The mass selective detector

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parameters were as follows: ms source 250 °C, ms quad 200 °C, SIM mode: target

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(quantifying) ion at m/z 107 and qualifier ion at m/z 220. 4-n-NP was identified based

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on comparison of the examined sample retention time to the reference standard

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retention time. The quantitative analyses were achieved using standard curves,

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which showed linearity in the ranges from 0 µg mL −1 to 100 µg mL−1 of 4-n-NP.

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2.6. Derivatization and metabolite identification

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4-n-NP metabolite identification was completed according to the modified methods

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described in our previous papers (Krupiński et al. 2013; Różalska et al. 2010). Briefly,

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200 µL of extract dissolved in ethyl acetate was evaporated to dryness under a N2

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gas stream. Next, 50 µL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) was

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added and heated to 60 °C for 1 h. Then, the samples were supplemented with 200

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µL ethyl acetate and analyzed with GC–MS scan mode while the mass range was set

116

from 45.0 amu to 550.0 amu. The injection volume was the same as described

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above, but the inlet was set to a splitless mode. All 4-n-NP biodegradation products

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were identified on the basis of the mass spectra analysis (and compared with

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standards wherever possible) completed with an Isotope Calculator (NIST) and

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AMDIS software, and they were confirmed by the NIST08 MS library (Krupiński et al.

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2013; Różalska et al. 2010; Słaba et al. 2013b).

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3. Results and Discussion:

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3.1. Identification of fungal isolate IM 6519

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The fungal strain IM 6519 forms white colonies on PDA medium. After 8-10 days,

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when conidia begin to form, the colonies become green and the culture pigmentation

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becomes pale olivine. Mature colonies are slightly fluffy and low, with a visible yellow

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pigmentation sphere around the colonies, reaching 6 cm – 6.5 cm in diameter after

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10 days at 28 °C. Conidia are 2.5 µm – 3.5 µm x 6 µm – 7.5 µm, and the phialides

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are 2 µm – 3 µm x 10 µm – 17.5 µm. Despite the morphological observations, to

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identify strain IM 6519, a phylogenetic analysis based on ITS sequences (accession

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numbers JX438656) was performed. The results revealed that strain IM 6519

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clustered in the genus of Metarhizium. To properly classify this strain to the species

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rank, a multigene approach was completed (Kepler and Rehner 2013). The gene

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sequences obtained for TEF, MzIGS3, MzIGS2 and MzFg543 were deposited in

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GenBank under accession numbers JX438649, KF423436, KF423434, and

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KF423432, respectively. The phylogenetic analysis based on the multigene

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approach, with comparison to other Metarhizium species, revealed that strain IM

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6519 is identical to Metarhizium robertsii (Fig. 1). Based on the above results, fungal

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strain IM 6519 was identified as Metarhizium robertsii.

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3.2. 4-n-NP utilization by M. robertsii IM 6519

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The degradation of the xenoestrogen by M. robertsii IM 6519 cultures is presented in

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Fig. 2. While in abiotic samples, a recovery of 95% - 98% of the applied xenobiotic

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was determined, and an analysis of the extracts inoculated with the tested strain

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showed a rapid decrease in the amount of 4-n-NP. After 24 h of incubation, only

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5.8% and 4.7% of 4-n-NP, added at the initial concentrations of 10 mg L−1 and 20 mg

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L−1, were detected in the culture extracts. At the end of the experiment, the amount of

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the xenobiotic in those cultures was lower than the detection limits. The utilization

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kinetics of the 40 mg L-1 of 4-n-NP was also very efficient; however, after one day of

149

incubation, more than 85% of the xenobiotic was degraded. The final amounts of 4-n-

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NP detected in those samples were below 0.8%.

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Microbial metabolism plays a major role in the elimination of NP in the natural

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environment. The microbial utilization of this EDC was reported for bacteria (Iwaki et

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al. 2014; Watanabe et al. 2012), Chlorella vulgaris (Gao et al. 2011) and several

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fungal species (Cajthaml et al. 2009; Krupinski et al. 2013). However, there are only

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a few reports indicating whether the degradation process is complete (Dubroca et al.

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2005; Krupinski et al. 2013). In this work, the ability of M. robertsii IM 6519 to

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mineralize 4-n-NP (Table 1) was determined. During the experiments with [ring-

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14

C(U)]-labeled 4-n-NP, the total recovery of the applied radioactivity was higher than

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96%, and after 96 h of incubation, the liberation of 14CO2 in M. robertsii cultures was

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observed. The mineralization rate was high, and the amount of liberated 14CO2

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exceeded 38%. The evolution of 14CO2 during NP degradation described for other

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filamentous fungi was lower than presented in this paper. Trametes versicolor

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mineralized 6% of the 4-n-NP after 12 d, and in A. versicolor cultures, 20% of the

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initially applied radioactivity was liberated to 14CO2 after 3 d of incubation (Dubroca et

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al. 2005; Krupinski et al. 2013). The data obtained in this work indicated that the M. 8

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robertsii IM 6519 4-n-NP degradation process is complete and results in the cleavage

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of the aromatic ring.

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3.3. Effect of 4-n-NP on M. robertsii IM 6519 growth

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M. robertsii IM 6519 growth in a liquid mineral medium supplemented with 4-n-NP is

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illustrated in Fig. 3. The most significant differences between the controls and the

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samples with 4-n-NP were observed at 24 h of incubation, while 40% growth

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inhibition (in the presence of 40 mg L-1 of 4-n-NP) was observed during the same

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period. From 48 h until the end of the experiments, the differences in biomass

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amount between samples with 4-n-NP and controls were less visible; at the end of

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the incubation period, the final biomass yield was even higher in samples with 4-n-NP

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in comparison with controls (Table 2).

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3.4 Qualitative analysis of 4-n-NP metabolites

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The samples for qualitative analysis of the degradation of metabolites formed during

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4-n-NP degradation by M. robertsii IM 6519 were collected at 6-h incubation intervals

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up to 72 h of incubation. After this time, 4-n-NP derivatives were not detected. The

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corresponding controls (biotic and abiotic) serving as references for 4-n-NP

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metabolite searching were also examined. In abiotic samples, BSFTA-derivatized 4-

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n-NP underwent a characteristic fragmentation pattern with three major ions: 292 m/z

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- molecular ion (M), 179 m/z - base peak CH2-Ar(aromatic ring)-O-TMS and 73 m/z

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(TMS), as precisely described in our previous studies (Krupiński et al. 2013;

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Różalska et al. 2010). For the first step 4-n-NP metabolite identification, the extracted

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ion mode with 73 m/z and 179 m/z was completed. Several detected metabolites

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possessed strong ion 267 m/z coming from the substructure of TMS–O–Ar (Aromatic

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ring)–CH2–O–TMS (Supplementary Fig. 1), and this additional parameter was added 9

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to extracted ion mode. The presence of the identified compounds was confirmed with

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the NIST08 Mass Spectra Database with a probability ranging from 90% to 99%. The

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mass spectra of all of the identified compounds are shown in Table 3, and the

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proposed structures of the selected metabolites with their interpretation are

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presented in Supplementary Fig. 1. Additionally, for all of the metabolites, the relative

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abundances were calculated, and the obtained data were subjected to principal

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component analysis (PCA with MarkerView software version 1.2.1. (AB Sciex, USA).

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Figure 4 shows the PC1/PC2 scores and loadings plots obtained by performing

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orthogonal rotation PCA, with normalization using total area sums and Pareto

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scaling. The major differences between samples with 4-n-NP and biotic controls

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occurred during the first 24 h of incubation. Between 24 h and 48 h of cultivation, the

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differences were still visible. At 72 h of incubation, samples with 4-n-NP were similar

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to controls, suggesting the completion of the degradation process. These data are

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concordant with our previous morphological observations, while after 3 d of

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incubation, the emergence of larger pellets with “hairy” morphology signified the

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completion of the removal process (Różalska et al. 2014). The location of the

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samples on the chart is most affected by the high amounts of 4-n-NP degradation

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metabolites such as 4-hydroxybenzoic acid (5), 4-(1-hydroxynonyl)phenol (20), 7-(4-

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hydroxyphenyl)heptanoic acid (27), 4-(8-hydroxynonyl)phenol (29) and 4-n-NP (13).

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The PCA analysis revealed that several metabolites are also present in control

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cultures. To elucidate whether these compounds are degradation products or

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naturally occurring in M. robertsii cells, the samples were subjected to further

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analysis. The obtained data show that 4-hydroxybenzoic acid (5), (3,4-

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dihydroxyphenyl)acetic acid (11), 2-(4-hydroxyphenyl)acetic acid (6), 1-(4-

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hydoxyphenyl)ethanone (2), 3-(3,4-dihydroxyphenyl) propanoic acid (15) and

10

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phenylacetic acid (1) originated from the 4-n-NP degradation pathway. 4-Hydroxy-3-

216

metoxybenzaldehyde (3), 4-hydroxy-3-metoxyacetophenone (4), 4-hydroxy-3-

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methoxybenzoic acid (8) and (4-hydroxy-3-methoxyphenyl)acetic acid (9) were also

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present in the control cultures and could not be regarded as degradation metabolites

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(crossed out in Fig. 4). To assume the PCA and dynamic analyses of the metabolite

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formation, the M. robertsii IM 6519 degradation pathway is described below.

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The proposed degradation pathway is complex. After 6 h of incubation, several

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degradation products with versatile hydroxylation in a 4-n-NP alkyl side-chain were

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detected (20, 26, 28, 29, 33, 35, 37, 38). The trend analysis of the nascent

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compounds revealed that they are present in samples with an incubation period of up

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to 24 h. The GC-MS analysis of TMS-derivatized samples showed four side-chain

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monohyroxylated compounds (20, 26, 28, 29) with molecular ion 380 m/z. The

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compounds 26, 28, 29 were identified on the basis of their mass spectra as follows:

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4-(6-hydroxynonyl)phenol (6’-OH-4-n-NP), 4-(7-hydroxynonyl)phenol (7’-OH-4-n-NP)

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and 4-(8-hydroxynonyl)phenol (8’-OH-4-n-NP). They showed the following

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fragmentation: 380 m/z – molecular ion (M), 290 m/z resulting from cleavage of TMS-

231

OH and 117 or 131 or 145 from the fragments of TMS-O-CH- R (R = CH3 or C2H5 or

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C3H7, respectively), indicating the position of the hydroxylation of the side chain.

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Those metabolites of 4-n-NP were not previously found in fungal cultures, but their

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presence with detailed mass spectra descriptions was previously published for plant

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cell cultures (Berger et al. 2005; Bokern and Harms 1997; Schmidt et al. 2004).

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Moreover, 8’-OH-4-n-NP (29) was also detected in the bile or liver of mosquito fish

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and Atlantic salmon (Thibaut et al. 2002). A similar degradation pattern (sub-terminal

238

oxidation) was described for n-alkanes, where a secondary alcohol is converted to

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the corresponding ketone. These products are hydrolyzed by esterases, generating 11

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an alcohol and a short-chain acid (Rojo 2009). The presence of 21 and 22 suggests

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that this product could be further converted, similar to n-alkanes.

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The last side-chain monohyroxylated compound 4-(1-hydroxynonyl)phenol (20)

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showed common fragment ions at m/z 380 – molecular ion (M), 267 m/z (TMS-O-Ar-

244

O-CH2-O-TMS), 73 m/z, and 179 m/z (Supplementary Fig. 1). The presence of this

245

biodegradation product was documented in human liver microsomes incubated with

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4-n-NP (Deng et al. 2010; Tezuka et al. 2007). Herein, 4-(1-hydroxynonyl)phenol (20)

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was further converted to 9-hydroxy-9-(4-hydroxyphenyl)nonanoic acid (39), which

248

was previously detected in fungal cultures (Różalska et al. 2010; Różalska et al.

249

2013). The same conversion was described for human liver microsomes (Deng et al.

250

2010), but M. robertsii IM 6519 is able to further degrade this compound through

251

oxidations to the carboxylic acids and the detachment of terminal carbons, leading to

252

the formation of 4-hydroxy-4-(4-hydroxyphenyl)butanoic acid (18), which is likely

253

converted to 4-hydroxybenzoic acid (5).

254

During an inspection of the 4-n-NP metabolites, side-chain dihydroxylated

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compounds (33, 35, 37) were detected. These compounds possessed a hydroxyl

256

group next to the aromatic ring in the nonyl-moiety, while the second hydroxylation

257

was versatile. The presence of a differently substituted dihydroxy-4-n-NP derivative

258

with different OH-group positions on the side chain of 4-n-NP was also described in

259

wheat cell suspension cultures. These results were contrary to this study because

260

none of the metabolites were hydroxylated next to the aromatic ring in the alkyl chain

261

moiety (Bokern and Harms 1997).

262

The most common mechanism of 4-n-NP biodegradation by living organisms is

263

consecutive oxidation of the terminal methyl group of the aliphatic chain, leading to

12

264

the formation of carboxylic acids coupled with terminal carbon removal (Krupinski et

265

al. 2013;Vallini et al. 2001). This mechanism is similar to aerobic pathways for the

266

degradation of n-alkanes by terminal oxidation (Rojo 2009). During the degradation

267

of M. robertsii IM 6519 4-n-NP, the same mechanism was also detected as one of the

268

degradation routes. This process begins with the terminal oxidation of 4-n-NP,

269

leading to the formation of 9-(4-hydroxyphenyl)nonanoic acid (36). The subsequent

270

oxidation with the shortening of the alkyl chain leads to formation of 4-

271

hydroxybenzioic acid (5), which is converted to 3,4-dihydroxybenzoic acid (10) with a

272

probable ring fission. However, several detected metabolites seem to be further

273

hydroxylated in the aromatic ring. The presence of those metabolites in M. robertsii

274

IM 6519 cultures was observed after 24 h of incubation, and the first detected

275

metabolite, 6-(3,4-dihydroxyphenyl)hexanoic acid (31), was probably converted from

276

6-(4-hydroxyphenyl)hexanoic acid (19). Aromatic ring hydroxylations were

277

subsequently observed until the formation of 3,4-dihydroxybenzoic acid (10) from 4-

278

hydroxybenzoic acid (5). Phenol moiety hydroxylation during 4-n-NP removal is also

279

one of the described degradation routes (Tezuka et al. 2007). Metabolites with

280

versatile aromatic ring hydroxylations were detected in Wistar rat tissues, human liver

281

microsomes and in tobacco cell cultures; however, most metabolites were sulfate,

282

metoxy or glucuronide conjugates (Berger et al. 2005; Deng et al. 2010; Zalko et al.

283

2003). Also in microorganisms, the aromatic compound degradation pathways yield

284

the formation of metabolites possessing two neighboring hydroxyl groups attached to

285

the aromatic ring, e.g., catechols or dihydroxybenzoic acids, which subsequently

286

undergo ring-cleavage and lead to the production of compounds that are the

287

constituents of the TCA cycle (Krupinski et al. 2013). The metabolite 3,4-

13

288

dihydroxybenzoic acid (10) formed during M. robertsii IM 6519 4-n-NP degradation is

289

the last formation before cleavage of the aromatic ring.

290

The results suggest that in 4-n-NP degradation by M. robertsii IM 6519, several

291

metabolic routes are involved. In this study, the existence of fast and versatile

292

hydroxylation in the aliphatic chain was confirmed in fungal cultures. These

293

processes have only been described in higher eukaryotes such as fish or plants

294

(Berger et al. 2005; Bokern and Harms 1997; Thibaut et al. 2002), but the great

295

advantage of M. robertsii IM 6519 is the mineralization of the substrate, which

296

provides the complete removal of NP from the environment and avoids the

297

accumulation of toxic metabolites.

298

4. Conclusions

299

The M. robertsii IM 6519 isolated from larva degraded and mineralized 4-n-NP. Its

300

mineralization capability is a great advantage and avoids toxic metabolites or

301

conjugate accumulation in the environment. The degradation process was confirmed

302

by the identification of multiple metabolites. Herein, the coexistence of several

303

degradation routes with versatile hydroxylation in different alkyl chain positions was

304

described.

305

Acknowledgement:

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This study was supported by a grant from the National Centre for Science in Cracow,

307

Poland, No. UMO-2011/01/B/NZ9/02898.

308

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326

2010. Evidence for the bioactivation of 4-nonylphenol to quinone methide and

327

ortho-benzoquinone metabolites in human liver microsomes. Chem. Res.

328

Toxicol. 23, 1617-1628.

329

8. Dubroca, J., Brault, A., Kollmann, A., Touton, I., Jolivalt, C., & Mougin, C.

330

2005, "Biotransformation of nonylphenol surfactants in soils amended with

331

contaminated sewage sludges.," In Environmental Chemistry, E. Lichtfouse, J.

332

Schwarzbauer, & D. Robert, eds., Springer, pp. 305-315.

15

333

9. Gao, Q.T., Wong, Y.S., Tam, N.F., 2011. Removal and biodegradation of

334

nonylphenol by immobilized Chlorella vulgaris. Bioresour. Technol. 102,

335

10230-10238.

336

10. Iwaki, H., Fujioka, M., Hasegawa, Y., 2014. Isolation and characterization of

337

marine nonylphenol-degrading bacteria and description of Pseudomaricurvus

338

alkylphenolicus gen. nov., sp. nov. Curr. Microbiol. 68, 167-173.

339

11. Kepler, R.M., Rehner, S.A., 2013. Genome-assisted development of nuclear

340

intergenic sequence markers for entomopathogenic fungi of the Metarhizium

341

anisopliae species complex. Mol. Ecol. Resour. 13, 210-217.

342

12. Krupiński, M., Szewczyk, R., Długoński, J., 2013. Detoxification and

343

elimination of xenoestrogen nonylphenol by the filamentous fungus Aspergillus

344

versicolor. Int. Biodeter. Biodegrad. 82, 59-66.

345

13. Ojeda, G., Patricio, J., Navajas, H., Comellas, L., Alcaniz, J.M., Ortiz, O.,

346

Marks, E., Natal-da-Luz, T., Sousa, J.P., 2013. Effects of nonylphenols on soil

347

microbial activity and water retention. Appl. Soil Ecol. 64, 77-83.

348 349 350

14. Rojo, F., 2009. Degradation of alkanes by bacteria. Environ. Microbiol. 11, 2477-2490. 15. Różalska, S., Glińska, S., Długoński, J., 2014. Metarhizium robertsii

351

morphological flexibility during nonylphenol removal. Int. Biodeter. Biodegrad.

352

95, 285-293.

353

16. Różalska, S., Pawłowska, J., Wrzosek, M., Tkaczuk, C., Długoński, J., 2013.

354

Utilization of 4-n-nonylphenol by Metarhizium sp. isolates. Acta Biochim. Pol.

355

60, 677-682.

16

356

17. Różalska, S., Szewczyk, R., Długoński, J., 2010. Biodegradation of 4-n-

357

nonylphenol by the non-ligninolytic filamentous fungus Gliocephalotrichum

358

simplex: a proposal of a metabolic pathway. J. Hazard. Mater. 180, 323-331.

359

18. Schmidt, B., Patti, H., Hommes, G., Schuphan, I., 2004. Metabolism of the

360

nonylphenol isomer [ring-U-14C]-4-(3',5'-dimethyl-3'-heptyl)-phenol by cell

361

suspension cultures of Agrostemma githago and soybean. J. Environ. Sci.

362

Health B. 39, 533-549.

363

19. Słaba, M., Bernat, P., Różalska, S., Nykiel, J., Długoński, J., 2013a.

364

Comparative study of metal induced phospholipid modifications in the heavy

365

metal tolerant filamentous fungus Paecilomyces marquandii and implications

366

for the fungal membrane integrity. Acta Biochim. Pol. 60, 695-700.

367

20. Słaba, M., Szewczyk, R., Piątek, M.A., Długoński, J., 2013b. Alachlor oxidation

368

by the filamentous fungus Paecilomyces marquandii. J. Hazard. Mater. 261,

369

443-450.

370

21. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008.

371

Nonylphenol in the environment: a critical review on occurrence, fate, toxicity

372

and treatment in wastewaters. Environ. Int. 34, 1033-1049.

373

22. Stasinakis, A.S., Gatidou, G., Mamais, D., Thomaidis, N.S., Lekkas, T.D.,

374

2008. Occurrence and fate of endocrine disrupters in Greek sewage treatment

375

plants. Water Res. 42, 1796-1804.

376

23. Tezuka, Y., Takahashi, K., Suzuki, T., Kitamura, S., Ohta, S., Nakamura, S.,

377

Mashino, T., 2007. Novel metabolic pathways of p-n-nonylphenol catalyzed by

378

cytochrome P450 and estrogen receptor binding activity of new metabolites. J.

379

Health Sci. 53, 552-561.

17

380

24. Thibaut, R., Debrauwer, L., Perdu, E., Goksoyr, A., Cravedi, J.P., Arukwe, A.,

381

2002. Regio-specific hydroxylation of nonylphenol and the involvement of

382

CYPK- nd CYP2M-like iso-enzymes in Atlantic salmon (Salmo salar). Aquat.

383

Toxicol. 56, 177-190.

384

25. Tuan, N.N., Lin, Y.W., Huang, S.L., 2013. Catabolism of 4-alkylphenols by

385

Acinetobacter sp. OP5: genetic organization of the oph gene cluster and

386

characterization of alkylcatechol 2, 3-dioxygenase. Bioresour. Technol. 131,

387

420-428.

388

26. Vallini, G., Frassinetti, S., D'Andrea, F., Catelani, G., Agnolucci, M., 2001.

389

Biodegradation of 4-(1-nonyl)phenol by axenic cultures of the yeast Candida

390

aquaetextoris: identification of microbial breakdown products and proposal of a

391

possible metabolic pathway. Int. Biodeter. Biodegrad. 47, 133-134.

392

27. Watanabe, W., Hori, Y., Nishimura, S., Takagi, A., Kikuchi, M., Sawai, J.,

393

2012. Bacterial degradation and reduction in the estrogen activity of 4-

394

nonylphenol. Biocontrol. Sci. 17, 143-147.

395

28. Wu, J., Wang, F., Gong, Y., Li, D., Sha, J., Huang, X., Han, X., 2009.

396

Proteomic analysis of changes induced by nonylphenol in Sprague-Dawley rat

397

Sertoli cells. Chem. Res. Toxicol. 22, 668-675.

398

29. Zalko, D., Costagliola, R., Dorio, C., Rathahao, E., Cravedi, J.P., 2003. In vivo

399

metabolic fate of the xeno-estrogen 4-n-nonylphenol in Wistar rats. Drug

400

Metab. Dispos. 31, 168-178.

401

18

402

Figure captions:

403

Fig. 1. Maximum-likelihood tree for the Metarhizium anisopliae complex. The strain

404

isolated during this study is in bold red. The bootstrap values are on the tree nodes.

405

Fig. 2. 4-n-NP elimination by M. robertsii IM 6519 in a mineral medium supplemented

406

with different xenobiotic concentrations.

407

Fig. 3. Time course of the growth of M robertsii IM 6519 in the presence of 4-n-NP at

408

concentrations of 10 mg L−1 (open squares), 20 mg L−1 (black triangles), and 40 mg

409

L−1 (open circles) compared to the control without substrate (black diamonds).

410

Supplementary Fig. 1. Mass spectrum analysis of selected 4-n-NP intermediates. (A)

411

4-(8-hydroxynonyl)phenol, di-TMS; (B) 8-(4-hydroxyphenyl)nonanal, TMS; (C) 4-(1-

412

hydroxynonyl)phenol, di-TMS.

413

Fig. 4. PCA of the M. robertsii IM 6519 metabolism in 4-n-NP-containing (40 mg L-1)

414

and control cultures on a mineral medium. On the left - PC1 against PC2 loadings

415

chart; on the right - PC1 against PC2 scores chart.

416

417

19

418

419

420

Fig. 1

421

422

20

423

424

425

Fig. 2

426

427

21

428

429

430

Fig. 3

431

432

433

434

22

435

436

437

Fig. 4

438

439

440

23

441

442

Table 1. Balancing of applied 14C radioactivity with 4-n-NP [ring-14C(U)]. Incubation time (h)

Filtrate

Biomass

CO2

Total recovery (%)

0

100

0

0

96.21 ± 3.18

96

61.60 ± 3.09

0.5 ± 0.08

38.15 ± 3.15

98.47 ± 1.15

Values of radioactivity are shown as % of the initial applied radioactivity.

443 444

445

24

446

Table 2. M. robertsii IM 6519 growth parameters in the presence of different

447

concentrations of 4-n-NP. 4-n-NP initial

Specific

Final biomass

amount

growth rate µ

yield (g L-1)

(mg L-1)

(h-1)

10

0.0239

9.07

20

0.0230

9.57

40

0.0244

8.68

Control (0)

0.0275

8.58

448

449 450

451

452

25

453 454

Table 3. GC-MS results of qualitative analysis of 4-n-NP degradation by M. robertsii IM 6519. ID

RT (min)

Compound name (TMS-derivatives)

Chemical formula

MW (Da)

1

7.520

Phenylacetic acid, TMS

C11H16O2Si

208.32

2

8.713

1-(4-hydroxyphenyl)ethanone, TMS

C11H16O2Si

208.33

3

9.134

4-hydroxy-3metoxybenzaldehyde, TMS

C11H16O3Si

224.32

4

9.641

4-hydroxy-3metoxyacetophenone, TMS

C12H18O3Si

238.35

5

9.652

4-Hydroxybenzoic acid, diTMS

C13H22O3Si2

282.49

6

9.716

2-(4-Hydroxyphenyl)acetic acid, di-TMS

C14H24O3Si2

296.52

7

10.411 3-(4Hydroxyphenyl)propanoic acid, di-TMS

C15H26O3Si2

310.54

8

10.432 4-hydroxy-3-methoxybenzoic acid, di-TMS

C14H24O4Si2

312.50

Mass spectrum m/z (10 largest ions relative intensity) 73 (99.9), 75 (34), 91 (19.7), 164 (19.7), 193 (12.5), 45 (9.9), 65 (9.9), 74 (9.7), 89 (5.8), 90 (5.7) 193 (99.9), 208 (25.6), 194 (16.3), 73 (14.6),151 (12.9), 45 (10.2), 75 (5), 89 (7.4), 91 (5.5),195 (4.8) 194 (99.9), 193 (48.7), 209 (46.1), 224 (31.6), 73 (21.7), 195 (16.3), 210 (7.5), 163 (6.4), 165 (6.3), 179 (6.0) 193 (99.9), 223 (98.9), 73 (70.8), 208 (52.2), 238 (42), 75 (19.1), 224 (17.8), 194 (17.1), 43 (15.8), 165 (10) 267 (99.9), 223 (73.6), 193 (58), 73 (50.9), 282 (24.4), 268 (23.5), 224 (16.4), 126 (11.8), 194 (10.3), 269 (9.8) 73 (99.9), 179 (30.1), 75 (16.1), 164 (15.8), 252 (15.7), 296 (915.3), 281 (14.7), 74 (9), 45 (8.5), 163 (6.6) 179 (99.9), 192 (63.3), 73 (29.3), 235 (24.6), 310 (22.4), 177 (17.4), 193 (16.9), 180 (15.6), 75 (13.1), 295 (7.2) 297 (99.9), 73 (76.4), 312 (67.2), 267 (64.4), 223 (53.2), 26

9

10.475 (4-hydroxy-3C15H26O4Si2 methoxyphenyl)acetic acid, diTMS

10 10.74

326.53

3,4-Dihydroxybenzoic acid, tri-TMS

C16H30O4Si3

370.67

11 10.798 (3,4-dihydroxyphenyl)acetic acid, tri-TMS

C17H32O4Si3

384.69

12 10.962 4-(4-hydroxyphenyl)butanoic acid, di-TMS

C16H28O3Si2

324.57

13 11.249 4-n-nonylphenol, TMS

C18H32OSi

292.54

14 11.346 (2E)-3-(4hydroxyphenyl)prop-2-enoic acid, di-TMS

C15H24O3Si2

308.52

15 11.395 3-(3,4dihydroxyphenyl)propanoic acid, tri-TMS

C18H34O4Si3

398.71

16 11.464 5-(4-hydroxyphenyl)pentanoic acid, di-TMS

C17H30O3Si2

338.60

253 (41.2), 282 (35.2), 298 (25.2), 126 (24.4), 193 (20.4) 73 (99.9), 209 (61.2), 326 (59.2), 179 (39), 311 (34.4), 267 (32.6), 75 (18.7), 327 (16.8), 296 (13.9), 210 (10.8) 193 (99.9), 370 (55.7), 73 (48.9), 355 (29.3), 311 (20.1), 371 (18.5), 194 (15.2), 281 (10.9), 223 (9.7), 356 (9.4) 73 (99.9), 179 (60.9), 384 (55.5), 267 (49.7), 385 (19.3), 237 (15.8), 75 (13.9), 268 (13.7), 45 (9.1), 369 (9.1) 192 (99.9), 73 (24.1), 193 (18.6), 179 (17.9), 309 (16.2), 324 (15.7), 177 (15.4), 75 (14.7), 147 (7.6), 194 (5.1) 179 (99.9) 292 (39) 180 (21.8) 73 (16.6), 293 (10.6) 181 (6.6) 277 (5.9) 163 (4.0), 165 (3.7) 149 (3.3) 293 (99.9), 219 (85.2), 308 (80), 73 (55.2), 249 (48.8), 294 (25.8), 309 (21.4), 220 (18.9), 179 (14.8), 250 (10.6) 179 (99.9), 398 (91.5), 267 (55.2), 73 (51.9), 399 (31.3), 280 (17.5), 180 (14.6) 400 (14.2), 268 (13.6), 383 (12.1) 179 (99.9), 73 (43.4), 75 (24.1), 338 (23.8), 192 (23.2), 323 (19.3), 180 (16.3), 149 (15.9), 205 (13.4), 206 (7.9) 27

17 11.858 4-(3,4dihydroxyphenyl)butanoic acid, tri-TMS

C19H36O4Si3

412.74

18 11.925 4-hydroxy-4-(4hydroxyphenyl)butanoic acid, tri-TMS

C19H36O4Si3

412.74

19 11.965 6-(4-hydroxyphenyl)hexanoic acid, di-TMS

C18H32O3Si2

352.63

20 12.127 4-(1-hydroxynonyl)phenol, diTMS

C21H40O2Si2

380.71

21 12.266 8-(4-hydroxyphenyl)nonanal, TMS

C18H30O2Si

306.51

22 12.237 7-(4-hydroxyphenyl)nonanal, TMS

C18H30O2Si

306.51

23 12.285 (2E)-3-(3,4dihydroxyphenyl)prop-2-enoic acid, tri-TMS

C18H32O4Si3

396.70

24 12.316 5-(3,4dihydroxyphenyl)pentanoic acid, tri-TMS

C20H38O4Si3

426.76

25 12.373 5-hydroxy-5-(4C20H38O4Si3 hydroxyphenyl)pentanoic acid, tri-TMS

426.76

412 (99.9), 280 (99.5), 73 (78.1), 267 (37.7), 413 (35), 235 (29.7), 281 (26.8), 179 (25.7), 397 (18.3), 75 (18) 412 (99.9), 73 (81.2), 267 (60.4), 179 (47.9), 413 (37.2), 268 (16.3), 414 (16.1), 233 (15.5), 75 (10.3), 74 (7.2) 179 (99.9) 352 (31.7) 73 (31) 180 (16.1) 337 (14.9) 75 (14.7), 353 (9.4) 253 (6.9) 181 (4.6), 177 (4.5) 380 (99.9), 267 (82), 73 (76.7), 179 (37.5), 381 (34.8), 268 (23.3), 382 (12.8), 269 (8.9), 365 (7.9), 180 (6.9) 179 (99.9), 306 (26.2), 73 (22), 180 (16.4), 43 (8.8), 181 (7.5), 307 (6.6), 163 (3.8), 75 (3.7), 149 (3.5) 179 (99.9), 306 (26.4), 73 (23), 180 (16), 181 (7.5), 57 (7.3), 307 (6.5), 192 (5.7), 75 (4.1), 177 (4) 219 (99.9), 73 (83.7), 396 (75), 397 (27.1), 381 (19), 191 (17.3), 220 (17.2), 218 (15.2), 179 (14.9), 398 (12.3) 426 (99.9), 73 (94.2), 267 (77.7), 427 (37), 205 (31.7), 75 (23), 268 (21), 411 (20.9), 179 (20.3), 428 (16.9) 73 (99.9), 426 (80.6), 267 (51.4), 179 (47.9), 427 (31.7), 75 (19.9), 268 (15), 253 28

26 12.373 4-(6-hydroxynonyl)phenol, diTMS

C21H40O2Si2

380.71

27 12.416 7-(4-hydroxyphenyl)heptanoic acid, di-TMS

C19H34O3Si2

366.65

28 12.523 4-(7-hydroxynonyl)phenol, diTMS

C21H40O2Si2

380.71

29 12.588 4-(8-hydroxynonyl)phenol, diTMS

C21H40O2Si2

380.71

30 12.753 6-hydroxy-6-(4hydroxyphenyl)hexanoic acid, tri-TMS

C21H40O4Si3

440.79

31 12.796 6-(3,4dihydroxyphenyl)hexanoic acid, tri-TMS

C21H40O4Si3

440.79

32 12.903 (4-Hydroxyphenyl)octanoic acid, di-TMS

C20H36O3Si2

380.66

33 13.104 4-(1,6-dihydroxynonyl)phenol, C24H48O3Si3 tri-TMS

468.89

(14.9), 428 (14), 147 (6.1) 73 (99.9), 179 (90.2), 290 (67.8), 145 (58.9), 205 (57.1), 75 (31), 291 (17.2), 192 (15.5), 180 (15.1), 206 (11.5) 179 (99.9), 73 (41.9), 366 (28), 75 (17.6), 180 (16.2), 351 (10.4), 367 (8.8), 253 (6.6), 181 (5.1), 177 (5) 179 (99.9), 73 (85.7), 131 (84.2), 290 (51.8), 205 (34.4), 75 (24.7), 351 (20.5), 380 (19.6), 180 (16.5), 291 (13) 179 (99.9), 117 (97.3), 73 (76.1), 290 (30), 75 (27.8), 380 (26.2), 205 (19.2), 180 (16.8), 118 (10.7), 365 (9.2) 73 (99.9), 440 (59.3), 179 (49.8), 267 (45.4), 75 (29.4), 441 (22.9), 197 (14.9), 268 (12.6), 442 (10.1), 180 (7.9) 73 (99.9), 440 (99.7), 267 (80), 179 (46.6) 441 (40), 268 (21.6), 75 (20.1), 253 (18.8), 442 (18), 87 (14.4) 269 (8.3) 179 (99.9), 73 (59.2), 380 (28.7), 180 (16.7), 75 (12.4), 381 (9.9), 253 (8.2), 103 (6.8), 181 (6.5), 147 (6.1) 73 (99.9), 468 (32.5), 179 (29.1), 267 (28.3), 145 (27.2), 75 (20.7), 469 (13.7), 378 (12), 293 (10.3), 268 (8.3) 29

34 13.169 7-hydroxy-7-(4C22H42O4Si3 hydroxyphenyl)heptanoic acid, tri-TMS

454.82

35 13.247 4-(1,7-dihydroxynonyl)phenol, C24H48O3Si3 tri-TMS

468.89

36 13.283 9-(4-hydroxyphenyl)nonanoic acid, di-TMS

C21H38O3Si2

394.69

37 13.311 4-(1,8-dihydroxynonyl)phenol, C24H48O3Si3 tri-TMS

468.89

38 13.605 4-(1,5-dihydroxynonyl)phenol, C24H48O3Si3 tri-TMS

468.89

39 13.97

482.27

9-Hydroxy-9-(4hydroxyphenyl)nonanoic acid, tri-TMS

C24H46O4Si3

454 (99.9), 73 (81.5), 267 (73.6), 179 (57.6), 455 (39.4), 268 (20.4), 75 (19.5), 439 (19.5), 456 (17.9), 180 (9.3) 73 (99.9), 468 (57.7), 267 (41), 131 (40.7), 179 (28.7), 469 (23.7), 75 (19), 268 (10.9), 253 (10.4), 439 (10.3) 179 (99.9), 73 (41.9), 394 (28), 180 (16.5), 75 (16), 379 (11.5), 395 (9.2), 181 (5.2), 177 (4.6), 253 (4.6) 73 (99.9), 468 (75.3), 117 (59), 267 (48.6), 179 (32), 469 (31.9), 75 (22.7), 268 (21.8), 211 (18), 253 (15.1) 73 (99.9), 468 (76.7), 267 (41.8), 179 (35.9), 469 (31), 75 (25.3), 470 (14.5), 411 (12.3), 268 (11.6), 221 (10.6) 73 (99.9), 482 (88.9), 267 (54), 179 (49.4), 483 (36.9), 75 (22.8), 484 (18.1), 467 (18), 268 (14.9), 74 (9.2)

455 456

457

458

30

459

460

Highlights: •

nonylphenol.

461 462

•

The coexistence of parallel degradation pathways is characteristic for this strain.

463 464

The new strain Metarhizium robertsii isolated from larvae biodegrades

•

M. robertsii mineralized nonylphenol.

465

31