# Investigation into the potential of Bjerkandera adusta to biotransform the recalcitrant fluoroquinolone antibiotic norfloxacin

Imen Akrout, Karima Staita, Jean Armengaud, Mélodie Kielbasa, David Navarro, Annick Turbé-Doan, Julien Lambert, Anne Lomascolo, Craig B Faulds, Héla Zouari-Mechichi, et al.

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## To cite this version:

Imen Akrout, Karima Staita, Jean Armengaud, Mélodie Kielbasa, David Navarro, et al. Investigation into the potential of Bjerkandera adusta to biotransform the recalcitrant fluoroquinolone antibiotic norfloxacin. Environmental Technology and Innovation, 2025, 40, pp.104414. 10.1016/j.eti.2025.104414. hal-05291436

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ELSEVIER

Contents lists available at ScienceDirect

Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

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Investigation into the potential of *Bjerkandera adusta* to
biotransform the recalcitrant fluoroquinolone
antibiotic norfloxacin

Imen Akrout<sup>a,b</sup>, Karima Staita<sup>a,b</sup>, Jean Armengaud<sup>c</sup>, Mélodie Kielbasa<sup>c</sup>, David Navarro<sup>a,d</sup>, Annick Turbé-Doan<sup>a</sup>, Julien Lambert<sup>a</sup>, Anne Lomascolo<sup>a</sup>, Craig B. Faulds<sup>a</sup>, Héla Zouari-Mechichi<sup>b</sup>, Giuliano Sciara<sup>a</sup>, Abdulrahman H. Alessa<sup>e</sup>, Tahar Mechichi<sup>b,*</sup>, Eric Record<sup>a,*</sup>

ARTICLE INFO

Keywords:

Biotransformation

Fluoroquinolones

Bjerkandera adusta

Peroxidase

Culture media

Proteomics

A B S T R A C T

Fluoroquinolones are among the most widely used broad-spectrum antibiotics worldwide. Due to inefficient removal by wastewater treatment plants, they accumulate in aquatic and terrestrial environments. Their persistence is concerning because it promotes the emergence of multidrug-resistant bacteria. This is alarming since the World Health Organization (WHO) classifies fluoroquinolones as critically important antibiotics, given the limited treatment options for severe bacterial infections in humans. Additionally, their accumulation can have toxic effects on various organisms. Previous studies reported the potential of ligninolytic fungi in transforming fluoroquinolones. In particular, a strain of *Bjerkandera adusta* (TM11), isolated in Tunisia, showed promising potential for biotransforming a broad spectrum of fluoroquinolones. Interestingly, *B. adusta* exhibited no laccase activity but peroxidase activity. Here we investigated the impact of culture medium composition on the strain's peroxidase activity and ability to biotransform norfloxacin, used as a model fluoroquinolone. We performed proteomic analysis of the secretomes from *B. adusta* cultured in four media, with and without antibiotics, to identify enzymes potentially involved in biotransformation. This study demonstrated that medium composition significantly influenced both peroxidase activity and norfloxacin biotransformation. The highest biotransformation rates, together with elevated peroxidase activity, were observed in media containing corn steep solids and yeast extract as nitrogen source after 7 days. Proteomic analysis revealed that presence of the antibiotic in the media induced the secretion of specific heme-peroxidase enzymes. Among them, two manganese peroxidases (MnP2 and MnP4), known for

<footnote><sup>a</sup> Aix-Marseille Université, INRAE Biodiversité et Biotechnologie Fongiques, Marseille 13288, France</footnote>

<footnote><sup>b</sup> Université de Sfax, Ecole Nationale d'Ingénieurs de Sfax, Laboratoire de Biochimie et de Génie enzymatique des lipases, Sfax 3038, Tunisia</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, Département Médicaments et Technologies pour la Santé, CEA, INRAE, SPI, Bagnols-sur-Cèze 30200, France</footnote>

<footnote><sup>d</sup> INRAE, Aix-Marseille Université, CIRMCF, Centre International des Ressources Microbiennes-Champignons Filamenteux, Marseille, France</footnote>

<footnote><sup>e</sup> Department of Biology, Faculty of Science, University of Tabuk, Tabuk 47512, Saudi Arabia</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: imen.akrout@etu.univ-amu.fr (I. Akrout), karima.staita@enis.tn (K. Staita), jean.armengaud@cea.fr (J. Armengaud), melodie.kielbasa@cea.fr (M. Kielbasa), david.navarro@inrae.fr (D. Navarro), annick.doan@univ-amu.fr (A. Turbé-Doan), julien.lambert@inrae.fr (J. Lambert), anne.lomascolo@univ-amu.fr (A. Lomascolo), craig.faulds@univ-amu.fr (C.B. Faulds), hela.zouari@isbs.usf.tn (H. Zouari-Mechichi), giuliano.sciara@inrae.fr (G. Sciara), tahar.mechichi@enis.rnu.tn (T. Mechichi), eric.record@inrae.fr (E. Record).</footnote>
their ligninolytic activity, emerged as particularly promising candidates for future studies on the enzymatic biotransformation of fluoroquinolones.

# 1. Introduction

Water scarcity is an escalating global issue, particularly in major urban areas, where shortages affected over a third of the population in 2016 rising to half by 2050. This crisis is exacerbated by climate change, rapid urbanization, and socio-economic trends (Distefano and Kelly, 2017; He et al., 2021). In addition, discharges of untreated or under-treated wastewater into natural water systems further deplete the already strained resources (Van Vliet et al., 2021). Among the various contaminants that pose challenges for water treatment are fluoroquinolones (FQs), a class of antibiotics widely used in human and veterinary medicine. Due to their low metabolism rate and high chemical stability, around 70 % of administered FQs are excreted unchanged into the environment via domestic and hospital wastewater, as well as effluent from livestock farms (Van Doorslaer et al., 2014). Conventional wastewater treatment plants often fail to remove these persistent compounds (Verlicchi et al., 2012). Indeed, the removal efficiency of FQs varies from 45 % to 77 %, depending on the antibiotic. As a result, a significant proportion of these residues remain in treated wastewater effluent (Verlicchi et al., 2012). Consequently, several FQs—such as norfloxacin, ciprofloxacin, enrofloxacin, and ofloxacin—are frequently detected in environmental waters (Chow et al., 2021; Van Doorslaer et al., 2014).

The environmental presence of FQs raises several concerns, primarily due to their high persistence. These compounds are poorly biodegradable by environmental microorganisms and exhibit a high affinity for solid matrices, protecting them from photodegradation (Du et al., 2023; Junge et al., 2011; Sukul and Spiteller, 2007). Furthermore, their low volatility and moderate water solubility facilitate their accumulation and mobility within water column (Bhatt et al., 2021; Van Doorslaer et al., 2014). Another major concern linked to the presence of FQs in the environment is their substantial contribution to the emergence of multi-resistant antibiotic bacteria. Numerous FQs-resistant bacterial strains have been reported over recent decades (Boni et al., 2022; Yang et al., 2020). This situation is particularly alarming, as the World Health Organization (WHO) classifies FQs as critically important antibiotics, due to the limited availability of alternative treatments for serious bacterial infections in humans (World Health Organization, 2019). Beyond antimicrobial resistance, the environmental accumulation of FQs poses ecological risks. Several studies have demonstrated their phytotoxic effects, as well as toxicity and bioaccumulation in aquatic organisms (Thai et al., 2023; Riaz et al., 2017; Bartoskova et al., 2014; Liu et al., 2014).

These challenges create an urgent need to find innovative wastewater treatment solutions for FQs. In this context, numerous studies have explored various FQs degradation approaches, including physical methods such as adsorption. Various solid matrices, like clay-based compounds, activated carbon and nanomaterials, have demonstrated effective FQs retention (Masud et al., 2024; Wan et al., 2013). However, limitations related to the regeneration and disposal of spent adsorbents hinder their practical applications and further investigations are required (Bhatt and Chatterjee, 2022). Some chemical degradation methods, notably advanced oxidation processes (AOPs), have also proven effective against FQs such as ciprofloxacin and moxifloxacin (Wang et al., 2019; Mi et al., 2019; Malesic-Eleftheriadou et al., 2019). Nevertheless, despite their oxidative potential and broad-spectrum reactivity, AOPs face practical limitations, including high operational costs, sensitivity to environmental parameters (e.g., pH, temperature), and difficulties in eliminating residual oxidants (Wang and Zhuan, 2020).

As a more cost-effective and environmentally sustainable alternative, biodegradation by microorganisms, particularly ligninolytic fungi, holds promising potential as agents for bioremediation. These fungi have emerged as promising candidates for the bioremediation of micropollutants, as they are equipped to secrete potent enzymes, such as laccases and peroxidases initially evolved for the degradation of lignocellulosic biomass (Akerman-Sanchez and Rojas-Jimenez, 2021; Rudakiya et al., 2019).

Previous studies have highlighted the potential of certain ligninolytic fungi to biotransform FQs (Ben Ayed et al., 2024, 2022; Prieto et al., 2011), and we recently demonstrated that a highly promising strain of *Bjerkandera adusta* achieved complete biotransformation of 8 out of 10 tested FQs commonly detected in wastewater, after a 15-day cultivation period underscoring the potential of this fungi for FQs bioremediation (Akrout et al., 2024).

Interestingly, *B. adusta* showed no laccase activity but displayed significant peroxidase activity, suggesting that peroxidases played a key role in its ability to biotransform FQs. Extracellular fungal peroxidases—classified as class II heme peroxidases—are secreted by ligninolytic fungi and include several types such as lignin peroxidases (LiP), manganese peroxidases (MnP), versatile peroxidases (VP), and dye-decolorizing peroxidases (DyP) (Sellami et al., 2022; Martínez, 2002). These enzymes are oxidoreductases that use hydrogen peroxide ($H_2O_2$) as an electron acceptor and heme as a redox cofactor to catalyze the oxidation of a wide range of organic and inorganic substrates (Zámocký et al., 2015; Banci, 1997). Due to their broad substrate specificity and high redox potential (average $E°$ ≈ +1140 mV), these enzymes are particularly valuable for biotechnological applications especially bioremediation (Khan et al., 2023; Barber-Zucker et al., 2022). They are capable of degrading persistent compounds through diverse reaction mechanisms, including hydroxylation, aromatic ring cleavage, and the breaking of C–C or C–O bonds (Sellami et al., 2022). As such, they have been reported to degrade a range of micropollutants, including industrial dyes (Chang et al., 2021), and pharmaceuticals such as antibiotics. For instance, Wen et al. (2009) demonstrated that a lignin peroxidase (LiP) purified from *Phanerochaete chrysosporium* biotransformed two antibiotics, tetracycline and oxytetracycline, with 95 % efficiency. However, cultivation parameters influence the diversity and quantity of enzymes—particularly peroxidases—secreted by fungi (Ambatkar et al., 2022; Vrsanska et al., 2015; Mester and Field, 2006; Mikiashvili et al., 2006), which could limit the application of these fungi in large-scale biotechnological processes such as the biotransformation of FQs. Although several studies have demonstrated the potential of certain ligninolytic fungi to biotransform FQs
(Ben Ayed et al., 2024; Čvančarová et al., 2015; Prieto et al., 2011), none have investigated how optimizing the composition of the culture medium could influence the enzymatic secretion profile and, consequently, affect the efficiency of FQs transformation. In this study, we evaluated the impact of culture medium composition on the peroxidase activity of *B. adusta* and its ability to biotransform norfloxacin, used here as a representative of fluoroquinolone antibiotics.

To gain deeper insight into the enzymatic mechanisms involved in FQs transformation, we performed proteomic analyses of the different fungal secretomes obtained under the different culture conditions with and without antibiotic. This comparative analysis provided a valuable basis for identifying candidate peroxidases potentially involved in FQs biotransformation. Overall, this study provides valuable knowledge to guide future research aimed at optimizing FQs transformation by ligninolytic fungi and elucidating the enzymatic mechanisms involved, with a view to application in bioremediation.

## 2. Materials and methods

### 2.1. Chemicals and fungus

All chemicals, unless otherwise specified, and the antibiotic norfloxacin were purchased from Sigma-Aldrich (Saint-Quentin-Fal-lavier, France). Bjerkandera adusta strain TM11 (NCBI accession number: ON340793) was isolated in northwest Tunisia (Daassi et al., 2016).

### 2.2. Media and preculture conditions

The fungal strain was maintained in solid plates composed of potato dextrose agar medium (39 g.L⁻¹), and liquid pre-cultures were prepared as described in Akrout et al. (2024). Briefly, precultures were cultivated in 250-mL baffled flasks containing 50 mL of a medium composed of malt extract (18 g.L⁻¹) and yeast extract (5 g.L⁻¹). The cultures were incubated at 30°C for 5 days with continuous stirring at 120 rpm.

### 2.3. Fungal culture conditions

All cultures and controls were incubated at 30°C on a rotary shaker at 120 rpm for 15 days. Erlenmeyer flasks containing 50 mL of the test media were used. The control medium was the basal medium (M7), composed of (per liter): glucose (10 g), peptone (5 g), yeast extract (1 g), ammonium tartrate (2 g), KH₂PO₄ (1 g), MgSO₄·7 H₂O (0.5 g), KCl (0.5 g), and a trace element solution (1 mL). The trace element solution consisted of 0.1 g. L⁻¹ Na₂B₄O₇·10 H₂O, 0.01 g. L⁻¹ CuSO₄·5 H₂O, 0.05 g. L⁻¹ FeSO₄·7 H₂O, 0.01 g. L⁻¹ MnSO₄·7 H₂O, 0.07 g. L⁻¹ ZnSO₄·7 H₂O, and 0.01 g. L⁻¹ (NH₄)₆Mo₇O₂₄·4 H₂O. The cultures were supplemented with norfloxacin at a final concentration of 50 mg. L⁻¹ and 300 µM CuSO₄.

This study examined three factors of effect of basal (M7) medium composition on peroxidase activity and biotransformation of norfloxacin: (i) the effect of adding peroxidase inducers, (ii) the effect of different carbon sources, and (iii) the effect of different nitrogen sources.

Each factor was analyzed independently using a 'one-factor-at-a-time' (OFAT) experimental design (Berger et al., 2018), in which we modified or added only one factor at a time to the M7 culture medium while keeping all other factors constant. The "OFAT" approach was selected as a screening design to assess individual effects of each tested compounds (i.e., inducer, carbon sources and nitrogen sources) on peroxidase activity and norfloxacin transformation.

The effect of peroxidase inducers on enzymatic activity and biotransformation was investigated using the control medium supplemented with gluconic acid (5 mM), glycolic acid (5 mM), manganese sulfate (0.5 mM), veratryl alcohol (5 mM), or vanillic acid (5 mM). We also studied the influence of adding Tween 80 as biosurfactant at a concentration of 0.1 % (v/v) on peroxidase activity. The impact of carbon sources on peroxidase activity and antibiotic biotransformation was evaluated by replacing glucose in the control medium with carboxymethyl cellulose (10 g.L⁻¹), xylan from beechwood (10 g.L⁻¹), or glycerol (10 g.L⁻¹), while maintaining the basal composition of the other components in the M7 medium. To study the effect of nitrogen sources, we used the same standard glucose-containing medium (M7) but with all nitrogen sources removed and added only one organic or inorganic nitrogen source as sole nitrogen source. The organic nitrogen sources tested included casein (5 g.L⁻¹), soya peptone (5 g. L⁻¹), yeast extract (5 g. L⁻¹), and corn steep solids (5 g. L⁻¹). The inorganic nitrogen sources included sodium nitrate (5 g. L⁻¹), ammonium nitrate (5 g. L⁻¹), and ammonium tartrate (5 g. L⁻¹). All media were adjusted to pH 5.5 by adding a few drops of diluted HCl 1 N. Uninoculated media containing the antibiotic solution were prepared as a control. Each culture was performed in triplicate.

### 2.4. Evaluation of residual antibiotic activity

The residual antibacterial activity of the antibiotics was evaluated by growth inhibition against *E. coli* (ATCC25922) as described in Akrout et al. (2024). The assay was performed with the well diffusion method, using Mueller-Hinter agar (Merck, Darmstadt, Germany) as the culture medium. An exponential growth culture of *E. coli* with an optical density of 0.08-0.1 was applied using a sterile cotton swab to inoculate the plates. Two holes were aseptically cut, and each filled with 50 µL of the culture supernatant (containing the antibiotic) or the control (abiotic control). The plates were incubated for 20 h at 37°C, and the *E. coli* inhibition-zones diameters obtained post-incubation were measured by a ruler on the surface of the petri dish. Measurements were performed for each of the 3 biological replicates. The percentage decrease in antimicrobial activity was calculated, for each replicate, as follows:
$$ \text{decrease in antimicrobial activity\%} = \left( \frac{\text{DtC} - \text{DtS}}{\text{DtC}} \right) * 100 $$

where DtC is the inhibition-zone diameter of the control, and DtS is the inhibition-zone diameter of the culture supernatant with antibiotics.

## 2.5. HPLC analysis of norfloxacin

Residual antibiotic concentrations after 7 and 15 days were determined by UV-HPLC, utilizing a Waters Symmetry C18 column (4.6 × 100 mm, 3.5 µM). Solvent A composed of 0.05 % (v/v) phosphoric acid and 5 % acetonitrile was used as mobile phase (at a flowrate of 1 mL min⁻¹), and solvent B composed of acetonitrile 100 % was used for elution of the antibiotic in the column. The gradient elution program was as follows: 100 % A for 2 min, 0 % B to 100 % B over 15 min, and 100 % B for 3 min. Injection volume was 5 µL. Total run-time was 15 min. Column temperature was held at 40°C, and the antibiotics were detected using a UV detector at 220 nm. Measurements were performed for each of the 3 biological replicates.

## 2.6. Peroxidase-like activity assays

To investigate the impact of culture medium composition on enzyme secretion, we measured peroxidase activity at various times in culture, based on the method described by Ngo and Lenhoff (1980). Specifically, peroxidase activity was assessed through a reaction involving 3-methyl-2-benzothiazolinone hydrazone (MBTH, 1 mM) and 3-(dimethylamino)benzoic acid (DMAB, 50 mM) in the presence of Mn²⁺ (MnSO₄ • H₂O, 1 mM) and H₂O₂. All reagents were dissolved in sodium acetate buffer (50 mM, pH=5), and the working solutions consisted of 2.5 mL of 100 mM sodium lactate buffer (pH=4.5), 2.5 mL of 100 mM sodium succinate buffer (pH=4.5), 0.5 mL of DMAB solution, 0.5 mL of MBTH suspension, and 1 mL of MnSO₄ solution. The absorbance of the solution was measured at 529 nm using an absorbance microplate reader at 30°C every 1 min for 30 min. Each well in a clear, flat-bottomed 96-well PCR plate was added with 140 µL of the working solution and 50 µL of the supernatant. The reaction was initiated by adding 10 µL of 5 mM H₂O₂. Measurements were performed for each of the 3 biological replicates.

## 2.7. Determination of fungal dry weight

After a 15-day cultivation period, the cultures were stopped and filtered through a Miracloth filter. The collected mycelia were rinsed thoroughly and transferred to pre-dried aluminum containers that had been carefully pre-tared. The samples were then dried at 105°C for 24 h until constant weight was recorded. To prevent moisture absorption, the containers were cooled in a desiccator for 30 min prior to final weighing. The dry weights of the mycelia were determined by subtracting the previously recorded tare weights of the empty containers from the total weights measured after drying. Measurements were performed for each of the 3 biological replicates.

## 2.8. Preparation of B. adusta cultures for proteomic analysis

To identify the enzymes potentially involved in the biotransformation of norfloxacin by B. adusta, we ran proteomic analysis on culture supernatants. These supernatants were collected from B. adusta cultures grown under three distinct conditions (as described in Section 2.3):

1. Cultivation in M7 medium with corn steep solids as nitrogen source

2. Cultivation in M7 medium using beechwood xylan as carbon source,

3. Cultivation in M7 medium supplemented with MnSO₄ as an inducer.

For each condition, two cultures with norfloxacin (50 mg.L⁻¹) and two cultures without norfloxacin were prepared. All cultures were incubated at 30°C for 7 days.

On the 7th day of culture, the supernatants were collected and filtered on a Miracloth membrane and centrifuged at 8000 g for 10 min at 4 °C. The resulting supernatants were filtered and concentrated as previously described in Ben Ayed et al. (2022). Protein concentrations in the obtained secretome samples with or without norfloxacin were determined using the Bradford method, with bovine serum albumin (BSA) as standard (Bradford, 1976).

## 2.9. Proteomic analysis of B. adusta secretomes

Proteins were diluted with LDS3X buffer (Thermo Fisher Scientific, Waltham, MA) to a final concentration of 26.5 mM Tris-HCl, 35.25 mM TRIS base, 0.5 % LDS, 2.5 % glycerol, 0.13 mM EDTA, and then heated for 5 min at 99°C. A quantity of 20 µg of exo-proteomes was subjected to denaturing electrophoresis for 5 min on a NuPAGE 4 %–12 % gradient gel with 50 mM 2-(N-morpholino) ethane sulfonic acid, 50 mM Tris Base, 0.1 % sodium dodecyl sulfate (SDS), 1 mM ethylene diamine tetraacetic acid pH7.3 as running buffer. After electrophoresis, the gel was stained with SimplyBlue SafeStain (Thermo Fisher Scientific). Each proteome was extracted as a single polyacrylamide band, processed as described in Rubiano-Labrador et al. (2014), and then proteolyzed with trypsin Gold
(Promega, Madison, WI) in 50 mM NH₄HCO₃ in presence of ProteaseMax detergent (Promega). Then 8 µL of the 40 µL peptide mixture was injected into an Easy-Spray PepMap Neo capillary column (2 µm, 100 Å, length: 75 µm ID × 50 cm length; Thermo Fisher Scientific), washed, and then resolved with a 120-min acetonitrile gradient at a 0.2 µL min⁻¹ flowrate. The gradient for fractioning peptides was 5–25 % acetonitrile in 0.1 % formic acid in 90 min followed by 25–40 % in 15 min, operated with a Ultimate 3000 nanoRSLC UHPLC system (Thermo Fisher Scientific). The column was coupled to a Q-Exactive HF instrument (Thermo Fisher

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Effects of various inducers on the peroxidase-like activity of B. adusta cultured in the presence of norfloxacin, and its ability to transform the antibiotic. (A-B) Effect of different types of inducers on the peroxidase-like activity of the secretomes: (A) various carboxylic acids and veratryl alcohol (B) Tween 80 and MnSO₄, compared to the control medium M7. (C) Reduction in norfloxacin concentrations (measured by HPLC). (D) Reduction in antimicrobial activity of B. adusta secretomes (assessed by antibiogram). (E) Dry weight of B. adusta mycelium after 15 days of cultivation. Each datapoint represents the mean ± standard deviation from triplicate experiments. The reduction percentages for (C) and (D) are calculated relative to a control without fungi containing the antibiotic incubated under the same conditions. Abbreviations: Aver, veratryl alcohol; Aglu, gluconic acid; Agly, glycolic acid; Av, vanillic acid; T80, Tween 80.</figcaption></figure>
Scientific) in data-dependent acquisition mode for analysis of peptides by tandem mass spectrometry, using the same parameters as described in Grenga et al. (2022). A TOP20 strategy was applied to select the 20 most abundant precursor ions (2 + or 3 +) for fragmentation, using a 10 s dynamic exclusion. The isolation window was set at 2 m/z, and intensity threshold was 8.3E4. Resolutions were set at 60,000 for the full scan of peptide ions (MS) and 15,000 for the secondary fragments (MS/MS). MS/MS spectra were assigned to peptide sequences using the MASCOT Daemon 2.3.2 search engine (Matrix Science, Boston, MA), with the *B. adusta* annotated genome from JGI (downloaded on 2024/01/09) and published by Binder et al. (2013), comprising a total of 15,473 protein sequences.

The search parameters were trypsin as proteolytic enzyme with two possible miscleavages, tolerances of 5 ppm for the MS signals and 0.02 Da for MS/MS signals, oxidation of methionine and deamidation of glutamine and asparagine as possible variable modifications, carbamidomethylation of cysteine as fixed modification, and a peptide p-value below 0.01. A protein was considered validated when at least two different peptides were detected but with at least one recorded as specific in the whole database. The false discovery rate for protein identification was below 1 %, as verified with a reverse decoy database search. Proteomic comparison was performed per Gouveia et al. (2020) using spectral counts as proxy for the protein abundances. Measurements were performed for each biological duplicate.

## 3. Results

We previously demonstrated that *B. adusta* efficiently biotransforms FQs under non-optimized cultivation conditions (Akrout et al., 2024). Interestingly, *B. adusta* lacks laccase genes (https://mycocosm.jgi.doe.gov/Bjead1_1/Bjead1_1.home.html) but is a potent secretor of peroxidases. Here, we started by investigating the impact of culture medium composition on peroxidase activity and norfloxacin biotransformation. The M7 basal medium, employed in our previous work, was used as the reference medium throughout the study. Specifically, we examined three main factors of M7 medium composition: (i) the effect of adding peroxidase inducers, (ii) the effect of different carbon sources, and (iii) the effect of different nitrogen sources. Each of these factors was analyzed independently using an 'OFAT' design (see 2.3).

### 3.1. Effect of various inducers on peroxidase-like activity and norfloxacin biotransformation

To enhance the process of FQ biotransformation by *B. adusta*, we examined the influence of various inducers added into the culture medium on peroxidase-like activity and norfloxacin biotransformation. We selected MnSO₄, gluconic acid, glycolic acid, vanillic acid, and veratryl alcohol as potential inducers. In addition, we evaluated the effect of Tween 80 as a biosurfactant promoting the release of enzymes potentially adsorbed to the mycelium. The selected inducers and Tween 80 were chosen based on previous studies that reported their ability to stimulate peroxidase activity in ligninolytic fungi (Mester and Field, 2006; Novotný et al., 2004; Urek and Pazarlioglu, 2007; Vrsanska et al., 2015). Cultures without any additives (M7 media) were used as controls. Peroxidase-like activity in the presence of these different compounds was measured spectrophotometrically over several days of culture. The biotransformation of norfloxacin was monitored by measuring the residual concentration of the compound in the culture medium and by evaluating its residual antibacterial activity against *E. coli* at 7 and 15 days of culture. We observed a higher peroxidase-like activity when the strain was cultured in presence of gluconic acid ('Aglu', Fig. 1A) and MnSO₄ (Fig. 1B) compared to control medium (M7). Specifically, at day 7, optimal activities were 69.2 L⁻¹ in presence of gluconic acid and 71.6 L⁻¹ in presence of MnSO₄, representing 1.92- and 1.86-fold higher activities than control (37.1 U. L⁻¹ in M7 medium), respectively.

Tween 80 (T80) also had a positive effect on peroxidase activity during the early stages of culture: at day 4, there was a peak in peroxidase activity (54.65 U. L⁻¹) 4.7-fold higher than in the M7 medium (11.63 U. L⁻¹). However, the effect of T80 rapidly diminished after this peak, possibly due to its metabolic biotransformation by the fungi (Zhang and Cheung, 2011). Continuous addition of T80 could therefore more effectively sustain elevated peroxidase activity throughout the culture period. Furthermore, studies have shown that T80 promotes faster nutrient adsorption, particularly glucose, in certain ligninolytic fungi like *Pleurotus tuber-regium*, leading to a quicker depletion of glucose in the medium (Zhang and Cheung, 2011). In our study, this may explain the accelerated peroxidase production in T80 supplemented cultures in the early stage of culture, as these enzymes are secreted during fungal secondary metabolism—a process known to be regulated by carbon availability (Conesa et al., 2002).

To assess the impact of various inducers added to the culture medium on norfloxacin biotransformation, we measured the residual concentration of the antibiotic by HPLC (Fig. 1C) and evaluated the residual activity of the 7-day and 15-day-old secretomes against *E. coli* (Fig. 1D). At day 7, none of the cultures had managed to fully eliminate the antibiotic. The most effective cultures were those with T80, MnSO₄, and the control medium M7, which all decreased norfloxacin concentration by approximately 60%–70%. Some of the cultures showed no ('Aglu') or only minimal depletion of the antibiotic (glycolic acid, 'Agly', and vanillic acid, 'Av'). However, a different pattern emerged after 15 days of culture, with most of the cultures showing at least 85%–90% reduction in norfloxacin concentration. However, except for T80, none of the added 'inducers' could match the complete depletion achieved with the control medium (M7). Interestingly, some of the cultures that showed no or minimal effect on the antibiotic at day 7 showed a dramatic improvement by day 15 (i.e. Aglu and Agly). The results for residual antibiotic activity (Fig. 1D) showed that at day 7, toxicity against *E. coli* cells was maintained for most of the *B. adusta* culture supernatants and only slightly reduced for MnSO₄, T80 and M7 media, whereas at day 15 the same three culture supernatants had zero toxicity to *E. coli*. The desynchronization between the disappearance of norfloxacin from the culture medium and suppression of the antibiotic effect suggests a multi-step detoxification mechanism, with intermediate products that retain their activity. In the gluconic acid condition, despite a significant reduction in norfloxacin concentration after 15 days of cultivation (Fig. 1C), there was only a very slight reduction in antibiotic activity (3.3%, Fig. 1D). This
suggests the presence of different biotransformation products with high residual activity, likely indicating different biotransformation pathways. Finally, to evaluate the influence of each compound on fungal growth, we measured the dry mass after 15 days of culture. As shown in Fig. 1E, vanillic acid, veratryl alcohol and MnSO₄ led to a significant reduction in dry mass compared to the reference medium M7, reflecting poorer growth of B. adusta. Overall, we were unable to find a direct correlation between global peroxidase activity and norfloxacin biotransformation. Notably, the peroxidases induced in presence of MnSO₄ and gluconic acid do not appear to be the ones driving FQ biotransformation. The main conclusion on the inducers tested is that none of the selected inducers successfully enhanced antibiotic biotransformation, and that M7 culture remains the best condition for norfloxacin biotransformation and inactivation.

### 3.2. Effect of different carbon sources on peroxidase-like activity and norfloxacin biotransformation

To investigate the effect of different carbon sources, glucose was replaced in the reference medium (M7) with carboxymethyl-cellulose (CMC), xylan, and glycerol at a concentration of 10 g.L⁻¹. The selected carbon sources were chosen based on previous studies that reported their positive effect on peroxidase activity in some ligninolytic fungi (Buswell et al., 1984; Mikiashvili et al., 2005; Roch et al., 1989). Control cultures were prepared in parallel using the standard medium (M7). First, we observed that modifying the carbon source did not alter the general trend in peroxidase-like activity over the cultivation period (Fig. 2A), which increased until day 7 then decreased significantly at day 15. Only the maximum of the activity varied, with xylan showing the highest peroxidase-like activity. However, these variations may be partly attributed to differences in fungal growth under the respective conditions (Fig. 2B). Comparing both the reduction in norfloxacin concentration across the different cultures (Fig. 2C) and the corresponding decrease in antibiotic activity (Fig. 2D), the M7 and xylan cultures showed similar patterns (in terms of both kinetics and magnitude). Both

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Effect of different carbon sources on the peroxidase-like activity of B. adusta cultured in the presence of norfloxacin, and its ability to transform the antibiotic. (A) Peroxidase-like activity of the secretomes; (B) Mycelium dry weight after 15 days of cultivation; (C) Reduction in norfloxacin concentrations (measured by HPLC); (D) Reduction in antimicrobial activity of B. adusta secretomes (assessed by antibiogram). The reduction percentages for (C) and (D) are calculated relative to a control without fungi containing the antibiotic incubated under the same conditions. Each datapoint represents the mean ± standard deviation from triplicate experiments. Abbreviations: CMC, carboxymethyl cellulose; Xy, xylan; Gly, glycerol.</figcaption></figure>
exhibited complete or near-complete norfloxacin depletion and a total loss of antibiotic activity against *E. coli* in the supernatants by day 15. The glycerol culture achieved near-total biotransformation of norfloxacin (90 % at day 15) but only partially detoxified it, as the reduction in antibiotic activity was only 52.96 % at day 15. This suggests the formation of toxic alternative biotransformation products different from those in the other carbon source conditions. Finally, the CMC culture showed a slower rate of norfloxacin biotransformation, with no reduction at day 7 but near-complete biotransformation and complete abolition of antibiotic activity at day 15. This is consistent with the overall reduced growth when CMC is used as carbon source (Fig. 2B).

### 3.3. Effect of different nitrogen sources on peroxidase-like activity and norfloxacin biotransformation

To investigate the effects of different nitrogen sources, we selected: (i) mineral nitrogen sources, i.e. ammonium nitrate (AN), ammonium tartrate (TA), and sodium nitrate (SN), based on previous studies that demonstrated their positive impact on peroxidase-like activity in some ligninolytic fungi (Mikiashvili et al., 2006; Wang et al., 2008); and (ii) organic nitrogen sources, i.e. casein, yeast extract (YE), soya peptone (Pep), and corn steep solids (CS), chosen based on previous studies that demonstrated their ability to enhance peroxidase-like activity in some ligninolytic fungi (Mikiashvili et al., 2006; Mester et al., 1996). These elements were added as sole nitrogen source, replacing the original nitrogen source in the M7 medium. The different nitrogen sources had variable but marked effects on the parameters measured (Fig. 3), particularly on the peroxidase-like activity (Fig. 3A) in contrast to the relatively stable effect observed with different carbon sources (Fig. 2A). Two cultures, CS and YE, exhibited higher overall peroxidase-like activity compared to other media, with peaks of 48.5 U.L⁻¹ at day 7 for CS and 31.4 U.L⁻¹ at day 9 for YE (Fig. 3A). A more rapid antibiotic

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Effect of different nitrogen sources on the peroxidase-like activity of B. adusta cultured in the presence of norfloxacin, and its ability to degrade the antibiotic. (A) peroxidase-like activity of the secretomes; (B) mycelium dry weight after 15 days of cultivation, (C) reduction in norfloxacin concentrations (measured by HPLC); and (D) reduction in antimicrobial activity of B. adusta secretomes (assessed by antibiogram). The reduction percentages for (C) and (D) are calculated relative to a control without fungi containing the antibiotic incubated under the same conditions Each datapoint represents the mean ± standard deviation from triplicate experiments. Abbreviations: AN, ammonium nitrate; TA, ammonium tartrate; Pep, peptone; Cs, corn steep solids; YE, yeast extract; SN, sodium nitrate.</figcaption></figure>
elimination and activity loss were also observed, becoming fully innocuous by day 7 with CS and YE (Fig. 3C and Fig. 3D). In contrast, a similar effect was only observed by day 15 in M7 cultures (Fig. 1D and Fig. 2D). The peroxidases secreted in the CS and YE media may thus possibly promote norfloxacin transformation.

Overall, the use of organic nitrogen sources resulted in a better growth, with higher mycelial biomass (Fig. 3B) compared to cultures grown in the presence of mineral nitrogen sources. However, it is important to note that these organic nitrogen sources are rich in nutrients and provide more than just nitrogen to the micro-organisms. The deleterious effect of mineral nitrogen sources was particularly evident in AN cultures, where dry biomass remained below 0.5 g·L⁻¹ (Fig. 3B). Consistently these cultures displayed almost no peroxidase-like activity (Fig. 3A), and had almost no detectable impact on either the concentration (Fig. 3C), or activity (Fig. 3D) of the antibiotic. The TA-based culture showed a different pattern: norfloxacin was eliminated from the medium at day 7 (Fig. 3C) but the loss of antibiotic activity was not observed until day 15 (Fig. 3D). Similarly, in SN cultures, despite total elimination of the antibiotic at day 15, only a minimal reduction in antibiotic activity was recorded at this time. Here again, this suggests that the biotransformation of norfloxacin involved distinct pathways and, most likely, different enzymes that were influenced by composition of the medium.

### 3.4. Proteomic content of the fungal secretomes produced in different culture conditions

To better understand the fungal response to norfloxacin exposure and identify enzymes potentially involved in norfloxacin transformation, we performed proteomic analyses of the secretomes of *B. adusta* after 7 days of cultivation (i.e. marking peak peroxidase activity in all conditions) (Table S1). For this analysis, culture media were selected from experiments involving different nitrogen sources and carbon sources. We selected conditions that exhibited high peroxidase-like activity and significant biotransformation of norfloxacin, i.e. specifically the corn steep-based medium and the xylan-based medium. We also performed cultures with the control medium (M7) and with control medium supplemented with MnSO₄ as an inducer of peroxidase-like activity (MnSO₄ medium). MnSO₄ medium showed slightly lower biotransformation after 15 days compared to control medium but also showed very high peroxidase-like activity. This lower biotransformation of norfloxacin could be due to the induction of one or more peroxidases that exhibit little to no effect on norfloxacin. Consequently, this medium was selected to differentiate the enzymes involved in the transformation of fluoroquinolones by *B. adusta*. For the proteomic experiments, all cultures were produced in duplicate, with each condition tested in both presence and absence of norfloxacin, to assess its effect on enzyme secretion. Overall, the number of different proteins secreted by the fungus remained roughly constant regardless of the growth medium provided (Fig. 4A), and the presence of antibiotic in the growth medium led to a global decrease in total number of proteins detected in secretomes across all media (Fig. 4A). The proportions of each major enzyme class—proteases, amidases, lipases, esterases, and carbohydrate-active enzymes (CAZymes)—remained largely constant (Fig. 4A). As CAZymes were the predominant enzyme class in the secretomes and encompassed essential ligninolytic enzymes and potent oxidoreductases, including peroxidases, we focused our efforts on this group. There was no significant culture medium composition effect on the distribution of the different types of CAZymes, which were largely glycoside hydrolases (GHs) in all conditions (Fig. 4B). However, the addition of norfloxacin systematically induced a reduction in the number of the different CAZymes detected. GHs were the most affected, losing 50 % on average following exposure to the antibiotic, except in the MnSO₄ condition where the major decrease was in number of AA2 enzymes (class II lignin-modifying peroxidases), which decreased

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Protein content variations in the secretomes of *B* adusta cultured with (+) and without (-) norfloxacin in different media. (A) Distribution of the proteins detected across the main enzyme families; (B) distribution of detected CAZymes (*including two non-CAZyme families: EXPN and DYP). Data represents the proportion of proteins in a specific family divided by total number of proteins detected. Abbreviations: GH, glycoside hydrolases; CE, carbohydrate esterases; PL, polysaccharide lyases; EXPN, expansins; AA, auxiliary activities; DYP, dye-decolorizing peroxidases.</figcaption></figure>
from 15 to 8 (Table S2) after addition of the antibiotic.

The observed trends in terms of enzyme diversity were also analyzed using the spectral count (SC) of each CAZymes family, which is an indicator of the relative abundance in a given secretome (Fig. 5): the GHs had a significantly lower SC when norfloxacin was present in the culture media, whereas peroxidases (AA2s + DyPs) were significantly induced by norfloxacin under all conditions except MnSO4. Moreover, among the different culture media tested, the CS medium—which exhibited the highest norfloxacin biotransformation rate (Fig. 3)—also induced the highest peroxidase secretion in the presence of the antibiotic (Fig. 5). This finding strengthens our initial hypothesis that certain peroxidases may be involved in the antibiotic transformation process. We thus focused our efforts on finding peroxidases potentially involved in FQs transformation.

In-depth analysis of the secreted peroxidases revealed that the presence of antibiotics had no significant effect on total number of AA2s and DyPs detected across all conditions, except for the MnSO4 condition (Fig. 6). Note that the higher proportion of AA2s in Fig. 4B was not attributed to an increase in the number of AA2s but rather to a decrease in the diversity of GHs detected. AA2 encompasses various types of heme-peroxidases, i.e. versatile peroxidase (VP), lignin peroxidase (LiP), and manganese peroxidase (MnP). Analysis of protein counts found that LiPs were generally the most represented (Fig. 6), followed by DyPs. In terms of protein relative abundance (i.e. SC), heme-peroxidases ranked among the top 10 most abundant enzymes in each condition (Tables S3-S6), and includes VP1 (ID: 43095), DYP2 (ID: 72253), and MnP2 (ID: 118718).

To identify the heme-peroxidases potentially involved, we focused on those that were both highly abundant (based on SC) and significantly induced in the presence of the antibiotic (based on fold-change). Based on these criteria, 7 heme-peroxidases were identified (Fig. 7 and Table S7): a versatile peroxidase (VP1, ID: 43095), two lignin peroxidases (LiP 5: ID: 121664, Lip 9: ID: 269481), two manganese peroxidases (MnP2, ID: 118718; MnP4, ID: 306863), and two dye-decolorizing peroxidases (DyP8, ID: 287732; DyP2, ID: 72253). VP1 was the most abundant heme-peroxidase across all media, particularly in the corn steep (628 SC) and MnSO4 (556 SC) conditions, compared to 181 in the M7 control medium. In M7, the lower basal secretion of VP1 was offset by an induction effect triggered when the antibiotic was present, suggesting a potential role of VP1 in norfloxacin biotransformation. MnP2 and MnP4 were predominantly induced in the corn steep and xylan media. MnP2, in particular, was highly abundant in the corn steep medium: 7-fold higher levels than in M7 and 1.7-fold higher levels than in the xylan medium. A similar pattern was observed for MnP4, although its overall SC was lower. Taken together, these findings indicate that MnP2 and MnP4 may be key enzymes contributing to the superior biotransformation performance of B. adusta in the corn steep medium (Figure 7). In contrast, other heme-peroxidases are likely not involved in antibiotic transformation or only play a minor role. For example, MnP1 (ID: 43329) was highly abundant in the MnSO4 medium, both with and without antibiotic, but was absent in the M7 condition and only had very low counts in other the media with antibiotic (Figure S7). Even though MnSO4 medium showed the highest peroxidase activity, its norfloxacin transformation rate at day 7 was not higher than other media (Fig. 7), suggesting that MnP1 is not a key enzyme in norfloxacin biotransformation.

To provide an overview of the induction profiles of the differentially-expressed peroxidases, we generated a heat map to represent the fold changes after the addition of norfloxacin in the various culture media (Fig. 8). Interestingly, the corn steep medium, despite being the best-performing medium for norfloxacin transformation, did not show a strong induction profile for the selected peroxidases. Only MnP2 and MnP4 showed notable induction (see above). This can be attributed to the fact that most of these peroxidases were already highly induced by the corn steep medium and were secreted in substantial amounts even in absence of the antibiotic. In contrast, xylan and M7 media displayed stronger induction profiles, with several LiPs strongly induced by norfloxacin (e.g. LiP9, LiP10, and LiP3 in xylan, and LiP9, LiP5, and LiP4 in M7) along with MnP2 and MnP4. Overall, the MnSO4 medium showed minimal induction of these peroxidases in response to norfloxacin

It could be hypothesized that this variation in induction is due to the presence of similar regulatory factors influencing the secretion of these peroxidases in the presence of the antibiotic. Indeed, the proximity of certain genes encoding some of these peroxidases (Figure S8), on the genome of B. adusta, suggests a shared regulatory mechanism. However, this hypothesis was not verified for all the secreted peroxidases, and so further studies are required.

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Relative spectral counts for CAZyme families (including two non-CAZyme families: EXPN and DYP) in each secretome from cultures with (+) and without (-) norfloxacin. The spectral count is an indication of the abundance of a given protein or protein family. Abbreviations: GH, glycoside hydrolases; CE, carbohydrate esterases; PL, polysaccharide lyases; EXPN, expansins; AA, auxiliary activities; DYP, dye-decolorizing peroxidases.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Distribution of heme-peroxidases detected in the secretomes of B. adusta cultured with (+) and without (-) norfloxacin. Abbreviations: DyP, dye-decolorizing peroxidases; VP, versatile peroxidase; MnP, manganese peroxidases; LiP, lignin peroxidases.</figcaption></figure>

<table><thead><tr><th colspan="6">Lip9 (39665 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>177</td><td>191</td><td>1.1</td><td>0.44</td><td>100</td></tr><tr><td>Xylan</td><td>14.5</td><td>111.5</td><td>7.3</td><td>3.41</td><td>59.9</td></tr><tr><td>M7</td><td>89</td><td>800.5</td><td>8.9</td><td>2.02</td><td>57.1</td></tr><tr><td>MnSO4</td><td>27</td><td>7.5</td><td>0.3</td><td>0.11</td><td>63.1</td></tr></tbody></table>

<table><thead><tr><th colspan="6">Lip5 (38941 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>92</td><td>133.5</td><td>1.4</td><td>0.53</td><td>100</td></tr><tr><td>Xylan</td><td>39</td><td>93.5</td><td>2.4</td><td>0.80</td><td>59.9</td></tr><tr><td>M7</td><td>20.5</td><td>134</td><td>6.3</td><td>0.17</td><td>57.1</td></tr><tr><td>MnSO4</td><td>50</td><td>93</td><td>1.8</td><td>0.18</td><td>63.1</td></tr></tbody></table>

<table><thead><tr><th colspan="6">MnP2 (37961 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>224</td><td>637.5</td><td>2.8</td><td>0.87</td><td>100</td></tr><tr><td>Xylan</td><td>54.5</td><td>366</td><td>6.6</td><td>2.22</td><td>59.9</td></tr><tr><td>M7</td><td>23</td><td>89.5</td><td>3.8</td><td>0.52</td><td>57.1</td></tr><tr><td>MnSO4</td><td>55</td><td>37.5</td><td>0.7</td><td>0.18</td><td>63.1</td></tr></tbody></table>

<table><thead><tr><th colspan="6">MnP4 (38496 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>59</td><td>133</td><td>2.2</td><td>0.08</td><td>100</td></tr><tr><td>Xylan</td><td>9</td><td>73</td><td>7.4</td><td>2.18</td><td>59.9</td></tr><tr><td>M7</td><td>1</td><td>5.5</td><td>3.3</td><td>5.52</td><td>57.1</td></tr><tr><td>MnSO4</td><td>13</td><td>9</td><td>0.7</td><td>0.11</td><td>63.1</td></tr></tbody></table>

<table><thead><tr><th colspan="6">Dyp2 (53215 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>241</td><td>369</td><td>1.5</td><td>0.07</td><td>100</td></tr><tr><td>Xylan</td><td>46</td><td>191.5</td><td>4.1</td><td>1.35</td><td>59.9</td></tr><tr><td>M7</td><td>302.5</td><td>290.5</td><td>1.0</td><td>0.22</td><td>57.1</td></tr><tr><td>MnSO4</td><td>98</td><td>197</td><td>2.0</td><td>0.13</td><td>63.1</td></tr></tbody></table>

<table><thead><tr><th colspan="6">Dyp8 (53006 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>86.5</td><td>176</td><td>2.0</td><td>0.5</td><td>100</td></tr><tr><td>Xylan</td><td>32.5</td><td>45</td><td>1.4</td><td>0.35</td><td>59.9</td></tr><tr><td>M7</td><td>86.5</td><td>73</td><td>0.8</td><td>0.19</td><td>57.1</td></tr><tr><td>MnSO4</td><td>44</td><td>45</td><td>1.0</td><td>0.05</td><td>63.1</td></tr></tbody></table>

<table><caption>**Fig. 7.** Induction of peroxidases of interest in the secretomes of B. adusta cultured in different media with (AB+) or without (AB-) antibiotics. Induction was measured by comparing total spectral counts and calculating the corresponding fold-change. The spectral count is an indication of the abundance of a given protein. SD, standard deviation (each datapoint represents the result of duplicate biological experiments).</caption><thead><tr><th colspan="6">VP1 (38387 Da)</th></tr><tr><th>Conditions</th><th>AB-</th><th>AB+</th><th>Fold change</th><th>SD Fold change</th><th>(%) of transformation at 7 days</th></tr></thead><tbody><tr><td>Corn steep</td><td>628</td><td>757.5</td><td>1.2</td><td>0.44</td><td>100</td></tr><tr><td>Xylan</td><td>387.5</td><td>652</td><td>1.7</td><td>0.3</td><td>59.9</td></tr><tr><td>M7</td><td>181</td><td>423.55</td><td>2.3</td><td>0.47</td><td>57.1</td></tr><tr><td>MnSO4</td><td>556.5</td><td>740.5</td><td>1.3</td><td>0.08</td><td>63.1</td></tr></tbody></table>
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Heat map representing the fold-change of selected peroxidases after adding antibiotic. The color scale represents the fold-changes in total spectra counts detected for a given protein in a given secretome, cultured with or without antibiotic. The color scale ranges from white (0.3) to dark red (19.2).</figcaption></figure>

**4. Discussion**

This study aimed to investigate some of the parameters that influence peroxidase secretion, and the ability of *B. adusta* to transform norfloxacin, used here as a model fluoroquinolone. Analysis of the effect of culture medium composition relative to the reference medium M7 found that modifying the carbon source had limited effect on the overall trend of peroxidase-like activity. The only notable difference was slightly higher peroxidase-like activity in the medium containing xylan as a carbon source. Mikiashvili et al. (2006) also reported an increased peroxidase-like activity in two *P. ostreatus* strains when providing a polysaccharide carbon source such as xylan. This could be explained by the fact that xylan, as a complex carbon source, may reduce the repressive effect observed in the presence of glucose on the expression of certain enzymes, including peroxidases. This repression is thought to be mediated by carbon catabolite repression mechanisms involving transcriptional regulators such as CreA (Adnan et al., 2017). In support of this, Janusz et al., (2013) identified creA sites in the promoter regions of some peroxidase genes. However, although higher peroxidase-like activity was observed with xylan, the difference compared to the M7 medium remained modest. The weak effect observed here may be explained by the low recalcitrance of the carbon source provided. Providing more recalcitrant lignocellulosic carbon sources would likely trigger additional pathways of this ligninolytic fungi. The study by Mikiashvili et al., (2005) showed, for example, that the use of grapevine sawdust as a carbon source strongly stimulated peroxidase secretion in *T. versicolor*. However, we decided to restrict our selection to simple and soluble carbon sources in order to avoid matrix effects and continue in scalable conditions.

In contrast to carbon sources, modifying the nitrogen sources in the culture media led to substantial variations in peroxidase-like activity profiles among the different conditions tested. Several studies have demonstrated that the regulation and expression of genes encoding ligninolytic enzymes are controlled by nitrogen sources through the presence of two transcription factor binding sites, Nit2 and AP2, in the promoter regions of certain peroxidase-encoding genes (Janusz et al., 2013) This could explain the pronounced
differences in peroxidase activity profiles observed across the tested media. The highest peroxidase-like activity was obtained when using corn steep and yeast extract as nitrogen source, which correlated with a faster antibiotic transformation rate in these media (day 7) compared to M7 (day 15) and an induced secretion of peroxidases detected by proteomic analysis. In addition, transformation percentages were higher with organic nitrogen sources compared to inorganic sources. Interestingly, Mester et al. (1996) reported that organic nitrogen sources had a stimulatory effect on the growth of *B. adusta* BOS55 compared to inorganic sources.

These results clearly indicate that the addition of specific nitrogen sources directly influences peroxidase-like activities and FQs transformation efficiency. However, further investigations into C/N ratio would be valuable to fully optimize heme-peroxidase secretion and enhance FQs transformation by *B. adusta*. Indeed, several studies have highlighted C/N ratio as a critical factor in ligninolytic enzyme secretion. For example, Huang et al. (2020) demonstrated that LiP and MnP activities in *Phanerochaete chrysosporium* were higher in nitrogen-limited media with a high C/N ratio. Conversely, Robinson et al. (2001) found that heme-peroxidase activity in *B. adusta* and *Phlebia tremellosa* was higher in nitrogen-rich media than in nitrogen-poor media. This suggests that the correlation between culture medium composition and ligninolytic enzyme production is strain-dependent.

Overall, regarding norfloxacin transformation across different culture conditions, at day 7, HPLC often showed a significant decrease in norfloxacin concentration in several cultures whereas antibiotic activity against *E. coli* was only moderately reduced. Complete loss of antibiotic activity was observed at day 15 in most of the cultures. A possible explanation would be that the transformation of norfloxacin by *B. adusta* occurs in phases involving different enzymatic steps, and producing different transformation products over the course of time with varying degrees of antibiotic activity. Prieto et al. (2011) proposed multi-step mechanism for the transformation of norfloxacin for the ligninolytic fungi *T. versicolor*. Furthermore, other studies have investigated the metabolic pathways of some filamentous fungi exposed to various xenobiotics, some of which share structural similarities with FQs, such as some polycyclic aromatic hydrocarbons (PAHs) (Aranda, 2016; Sutherland, 1992) or some endocrine disruptors (Cajthaml, 2015). The metabolism of these compounds generally follows a three-phases process. Extracellular enzymes are primarily observed in phase I of the transformation, which involves the initial modification of the chemical structure, typically through oxidation, facilitating further transformation by intracellular enzymes. However, in certain cases, extracellular enzymes such as peroxidases can also play a role later in the transformation process by performing an additional oxidation of metabolites generated by intracellular enzymes (Harms et al., 2011). A similar mechanism could be envisioned for the transformation of norfloxacin by *B. adusta*, with peroxidases involved first in generating primary oxidized metabolites. Indeed, in studies investigating the transformation of various FQs containing a piperazine ring, such as norfloxacin, an initial opening of the piperazine ring is frequently observed (Cvančarová et al., 2015; Prieto et al., 2011; Parshikov et al., 2001). These transformation products are likely generated by oxidative enzymes, such as peroxidases. Deciphering the precise mechanisms at work would require performing analytical measurements at different timepoints on *B. adusta* cultures exposed to norfloxacin.

Proteomic data showed that addition of the antibiotic led to an overall decrease in total number of enzymes secreted by the fungi under all tested conditions, suggesting that the presence of the antibiotic, despite being tolerated by the fungus, induces a stress. Studies have reported a similar decrease in total number of enzymes for other types of stresses, for instance in the secretome of *Pestalotiopsis* sp. under salt stress conditions (Arfi et al., 2013). Analysis of CAZymes found that the addition of the antibiotic resulted in a reduced abundance of GHs whereas heme-peroxidases were comparatively strongly induced in most culture media. It is well known that some genes encoding fungal ligninolytic heme-peroxidase have xenobiotic response elements in their promoters (Fernández-Fueyo et al., 2014; Janusz et al., 2013), which could explain the inducer effect of the antibiotic on these enzymes, and which further underscores the value of targeting these enzymes for the remediation of certain xenobiotics. This observation strengthens our initial hypothesis that peroxidases may be involved in FQs transformation. However, not all peroxidases secreted by *B. adusta* appear to be involved in FQs transformation. For instance, even though MnSO₄ and Aglu strongly stimulated general peroxidase activity, they did not have better norfloxacin transformation. In this study, among all secreted peroxidases, we identified seven heme peroxidases that could potentially be involved in the transformation of FQs by *B. adusta*. These enzymes belong to all four different peroxidase classes (MnP, LiPs, VPs and DyPs). These findings align with the existing literature, as fungal MnPs and LiPs are recognized as highly efficient oxidoreductases and widely utilized in various biotechnological applications, particularly in the remediation of pollutants such as plastics, textile dyes, and certain antibiotic (Singh et al., 2024; Srikanth et al., 2022; Sun et al., 2021). In the case of DyPs, Staita et al. (2024) demonstrated previously that a DyP from *Corilopsis gallica* was capable of transforming certain FQs. Finally, a VP from *B. adusta* was identified in a previous study and shown to transform antibiotics such as sulfamethoxazole (Eibes et al., 2011). This enzyme may be the same as VP1, which we detected as the most abundant peroxidase across most culture media. Eibes et al. (2011). did not provide the full sequence of the VP they identified, except for its N-terminal region, which is identical to that of VP1. Additionally, *B. adusta* possesses only a single known VP gene in its genome. These findings strongly suggest that both enzymes correspond to the same VP. However, accurately determining the role of these enzymes in FQs transformation requires the identification of the metabolites produced by the action of these purified enzymes on these antibiotics.

This study also highlighted that the same antibiotic molecule does not have exactly the same inducer effect on the heme-peroxidases, an effect that also depends on the culture medium. For instance, LiP9 was strongly induced after addition of antibiotic in the control medium (M7), reaching almost 45% of all AA2-member spectral counts (VPs, MnPs and LiPs), but was not as strongly induced in other conditions. This suggests that fungal response to xenobiotics is regulated by factors other than the presence of the foreign molecule. Taking all these lines of evidence together, we find that fungal bioremediation of antibiotics hinges on better understanding the regulatory mechanisms underlying the secretion of ligninolytic enzymes in the presence of xenobiotics, and that *B. adusta* could usefully serve as a convenient model organism to conduct this type of research.
## 5. Conclusion

This study provides novel insights into the ability of *B. adusta* to efficiently biotransform fluoroquinolones, a class of antibiotics that are recalcitrant to biotransformation and that contribute to the emergence of antibiotic resistance. We find peroxidase activity and antibiotic biotransformation by *B. adusta* are significantly influenced by the composition of the culture medium, which points to a highly complex regulation of heme-peroxidase-encoding genes. We observed strong peroxidase activity and a higher percentage of norfloxacin biotransformation compared to control medium (basal medium M7) after 7 days both corn steep-based medium and yeast extract-based medium. Proteomic analysis of the secretomes from cultures grown in corn steep-based medium revealed strong induction of certain heme-peroxidases compared to other media, suggesting a potential role for these enzymes in the biotransformation of norfloxacin by *B. adusta*. Among these enzymes, two manganese peroxidases (MnPs), MnP2 and MnP4, were identified as the most promising candidates for fluoroquinolone biotransformation. These enzymes could serve as models for further investigation into enzymatic applications for fluoroquinolone bioremediation, including heterologous expression and testing in biotransformation processes, either in free or immobilized form.

## Funding

The authors thank the Tunisian Ministry of Higher Education and Scientific Research for providing financial support for the first author (I.A.). We also thank the INRAE TRANSFORM division for providing further financial support through the Associated International Laboratory ‘BioDeg’, and Campus France for providing funding through the PHC-Utique ‘FUNZYBio’ project (Campus France Code: 47572QD; CMCU Code: 22G0814). This research was also supported by the FUNZYBIO project (‘Fungal and enzymatic biotransformation of antibiotics: safe reuse of 348 livestock residues for agriculture’; ANR-23-P012-0002), which is part of the European Union-sponsored PRIMA Program.

## CRediT authorship contribution statement

**David Navarro:** Writing – review & editing, Methodology, Investigation, Data curation. **Annick Turbé-Doan:** Writing – original draft, Methodology, Data curation. **Abdulrahman H. Alessa:** Writing – review & editing, Methodology. **Imen Akrout:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Tahar Mechichi:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Karima Staita:** Writing – review & editing, Methodology, Investigation. **Eric Record:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Jean Armengaud:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Mélodie Kielbasa:** Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Anne Lomascolo:** Writing – review & editing, Methodology. **Craig B. Faulds:** Writing – review & editing, Methodology. **Héla Zouari-Mechichi:** Writing – review & editing, Project administration, Methodology, Funding acquisition. **Giuliano Sciara:** Writing – review & editing, Project administration, Funding acquisition. **Julien Lambert:** Writing – original draft, Methodology, Data curation.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eric Record reports financial support was provided by Campus France. Eric Record reports financial support was provided by European Commission. Eric Record reports financial support was provided by French National Institute for Agricultural Research INRAE. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.eti.2025.104414.

## Data availability

Data will be made available on request.

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