Joyce Álvarez-Barragán

Cristiana Cravo-Laureau

Lukas Y Wick

Robert Duran
email: robert.duran@univ-pau.fr

Fungi in PAH-contaminated marine sediments: cultivable diversity and tolerance capacity towards PAH

Keywords: peroxidase genes, fungal ITS, PAH-uptake, Alternaria, PAH-contamination

The cultivable fungal diversity from PAH-contaminated sediments was examined for the tolerance to polycyclic aromatic hydrocarbon (PAH). The 85 fungal strains, isolated in non-selective media, revealed a large diversity by ribosomal internal transcribed spacer (ITS) sequencing, even including possible new species. Most strains (64%) exhibited PAH-tolerance, indicating that sediments retain diverse cultivable PAH-tolerant fungi. The PAH-tolerance was linked neither to a specific taxon nor to the peroxidase genes (LiP, MnP and Lac). Examining the PAH-removal (degradation and/or sorption), Alternaria destruens F10.81 showed the best capacity with above 80% removal for phenanthrene, pyrene and fluoranthene, and around 65% for benzo[a]pyrene. A. destruens F10.81 internalized pyrene homogenously into the hyphae that contrasted with Fusarium pseudoygamai F5.76 in which PAH-vacuoles were observed but PAH removal was below 20%. Thus, our study paves the way for the exploitation of fungi in remediation strategies to mitigate the effect of PAH in coastal marine sediments.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are important pollutants threatening the marine environment due to their toxicity [START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF]. Although the more spectacular input of PAHs in marine environments is due to accidental oil spills, the main source remains on natural oil seeps [START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF]. PAHs accumulate in sediments because of their hydrophobicity constituting a chronic contamination [START_REF] Rothermich | Anaerobic, sulfate-dependent degradation of polycyclic aromatic hydrocarbons in petroleum-contaminated harbor sediment[END_REF].

Their fate in the environment depends on biotic and abiotic factors [START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF].

Many microorganisms including archaea, bacteria, algae and fungi are able to degrade PAHs [START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF][START_REF] Bordenave | Diversity of ring-hydroxylating dioxygenases in pristine and oil contaminated microbial mats at genomic and transcriptomic levels[END_REF]Germouche M'rassi et al., 2015;[START_REF] Haritash | Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review[END_REF]. In the last years, the interest on PAHs removal and biodegradation by fungi has increased [START_REF] Mineki | Degradation of Polyaromatic Hydrocarbons by Fungi Isolated from Soil in Japan[END_REF][START_REF] Morales | Biodegradation of benzo[α]pyrene, toluene, and formaldehyde from the gas phase by a consortium of Rhodococcus erythropolis and Fusarium solani[END_REF]. The fungal removal of PAHs consists in three main processes: two oxidation processes involving extracellular peroxidases (lignin peroxidase, manganese peroxidase and laccase; [START_REF] Chen | Identification of genes for lignin peroxidases and manganese peroxidases in ectomycorrhizal fungi[END_REF][START_REF] Scheel | Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds[END_REF], and membrane attached monooxygenases (cytochrome P450; [START_REF] Črešnar | Cytochrome P450 enzymes in the fungal kingdom[END_REF][START_REF] Syed | Genome-tofunction characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)[END_REF], and absorption and storage of PAHs in lipid vacuoles [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF]. The ability of fungi to use PAHs as sole carbon and energy sources has been described [START_REF] Rafin | Degradation of benzo[a]pyrene as sole carbon source by a non white rot fungus, Fusarium Solani[END_REF].

However, it has been reported that most of fungi require co-metabolism with another carbon source for PAH degradation [START_REF] Cerniglia | Microbial metabolism of pyrene[END_REF].

Fungi have been found in all marine habitats [START_REF] Orsi | Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces[END_REF], revealing their high diversity [START_REF] Jones | Marine fungi: some factors influencing biodiversity[END_REF]. Ascomycota and Basidiomycota are the main fungal phyla found in marine environments as described for soil ecosystems [START_REF] Clemente | Biodegradation of polycyclic aromatic hydrocarbons by soil fungi[END_REF][START_REF] Field | Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi[END_REF][START_REF] Godoy | Exploring the potential of fungi isolated from PAH-polluted soil as a source of xenobiotics-degrading fungi[END_REF][START_REF] Li | Biodegradation of aged polycyclic aromatic hydrocarbons (PAHs) by microbial consortia in soil and slurry phases[END_REF][START_REF] Mineki | Degradation of Polyaromatic Hydrocarbons by Fungi Isolated from Soil in Japan[END_REF][START_REF] Potin | Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by Cladosporium sphaerospermum isolated from an aged PAH contaminated soil[END_REF][START_REF] Valentín | Biodegradation of polycyclic aromatic hydrocarbons in forest and salt marsh soils by white-rot fungi[END_REF]. Although fungi of terrestrial origin have been isolated from marine ecosystems [START_REF] Li | Diversity of fungal isolates from three Hawaiian marine sponges[END_REF], recent molecular analysis revealed specific fungal sequences suggesting the existence of novel species of marine fungi [START_REF] Amend | Fungi in the Marine Environment: Open Questions and Unsolved Problems[END_REF][START_REF] Grossart | Aquatic fungi: targeting the forgotten in microbial ecology[END_REF]. Fungi isolated from marine habitats exhibit similar morphological characteristics to their terrestrial counterparts [START_REF] Méjanelle | Sterols of melanized fungi from hypersaline environments[END_REF]. However, they might possess particular properties to survive in marine environments [START_REF] Amend | Fungi in the Marine Environment: Open Questions and Unsolved Problems[END_REF], particularly in PAHs contaminated sediments [START_REF] Greco | Fungal characterisation of a contaminated marine environment: the case of the Port of Genoa (North-Western Italy)[END_REF]. Such properties, as salinity tolerance and the capacity to degrade and accumulate PAHs, less bioavailable due to adsorption solid materials, remain to be explored [START_REF] Bonugli-Santos | Marine-derived fungi: diversity of enzymes and biotechnological applications[END_REF][START_REF] Bugni | Marine-derived fungi: a chemically and biologically diverse group of microorganisms[END_REF][START_REF] Trincone | Potential biocatalysts originating from sea environments[END_REF].

This study aimed to explore the cultivable marine fungi, recovered from oil-contaminated saline sediments, for their PAH-tolerance capacity. For this purpose fungal strains were isolated from various marine coastal environments, characterized and identified by ITS sequence analysis, and their features explaining the PAH tolerance examined.

Material and Methods

Culture media

The culture media used in this study were based on the seawater minimal medium (swMM ;[START_REF] Brito | Characterization of hydrocarbonoclastic bacterial communities from mangrove sediments in Guanabara Bay, Brazil[END_REF], which composition was as follow: KCl 0.75 g/L, CaCl•2H2O 1.47 g/L, NH4Cl 1.5 g/L, MgSO4•7H2O 6.64 g/L, NaCl 20 g/L, Na2CO3 0.265 g/L, 1 mL of trace elements solution (H3BO3 300 mg/L, FeSO4•7H2O 1.1 g/L, CoCl2•6H2O 190 mg/L, MnCl2•2H2O 50 mg/L, ZnCl2 42 mg/L, NiCl2•6H2O 24 mg/L, Na2MoO4•2H2O 2mg/L), 1mL of vitamin solution (biotine 2 mg/L, ρ-aminobenzoate 10 mg/L, thiamine 10 mg/L, pantothenate 5 mg/L, pyridoxamine 50 mg/L, vitamin B12 20 mg/L, nicotinate 20 mg/L), and 100 µL of phosphate buffer 50 mM. The pH was adjusted with HCl to 6.5. Chemicals were purchased from Sigma Aldrich (Germany).

The malt dextrose agar (MDA) and malt dextrose (MD) media, in which distillated water was exchanged by swMM (MDAsw and MDsw respectively) to keep salinity conditions, were used for the isolation and for maintaining fungal strains.

Selection and conservation of fungal strains

Oil polluted sediment collected from different coastal areas were used as inoculum for the isolation of fungal strains with the ability to degrade PAHs. Each sample was inoculated directly in MDAsw and incubated for 5 days. Also, dilutions at 10 -1 , 10 -2 and 10 -3 were performed taking 100 mg of each source.

The isolated fungal strains were conserved as conidia and mycelia in glycerol at -70°C. Fungi were inoculated in MDsw grown until conidia overwhelmed cultures. Mycelia and conidia were recovered from the flask and then dispatched in at least 3 Eppendorf tubes (100 mg of biomass each) for each strain. After addition of 1 mL glycerol (30% solution), the tubes were frozen and kept at -70 °C until use. In order to check viability one tube with mycelia was tested after 7 days of storage by inoculating MDsw culture.

Fungi identification sequencing and phylogenetic analysis

Fungi were harvested from MDAsw cultures from 10 days of incubation and DNA was extracted using the QUIAGEN DNeasy® UltraClean® Microbial Kit (Cat. No. 12224-40) following the manufacturer instructions. The identification was based on ITS sequences, which were amplified using the primers ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) that amplify the ITS1, 5.8S and ITS2 region of the rRNA genes operon. The amplified region allows the identification at the species level and even at the subspecies level [START_REF] Fajarningsih | Internal transcribed spacer (ITS) as DNA barcoding to identify fungal species: a review[END_REF]. The PCR reaction mix was prepared with 1 µL of extracted DNA in 9.5 µL of DEPC-treated water, 1 µL of each primer (20 µM), 12.5 µL AmpliTaq Gold 360 Master Mix 2X (Thermo Fisher Scientific, USA).

The amplification was performed through 35 cycles of 95 °C (30 s), 55 °C (30 s) and 72 °C (1 min), with a previous activation start of 95 °C (10 min) and final extension step at 72 °C (10 min). ITS amplified fragments were sequenced at the Eurofins platform (France).

Sequence data were edited using Chromas Pro version 1.34. For identification, fungal ITS rRNA sequences were compared with NCBI (National Centre for Biotechnology Information; http://www.ncbi.nlm.nih.gov) database as previously described [START_REF] Giloteaux | Nested PCR and new primers for analysis of sulfate-reducing bacteria in low-cell-biomass environments[END_REF]. Fungal ITS sequences in this study and reference sequences from GenBank were edited and aligned using CLUSTAL-W [START_REF] Thompson | Multiple Sequence Alignment Using ClustalW and ClustalX[END_REF] as described [START_REF] Bruneel | Archaeal diversity in a Fe-As rich acid mine drainage at Carnoulès (France)[END_REF]. The aligned sequences were imported into MEGA 3.1 [START_REF] Kumar | MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment[END_REF] for creating Neighbour-joining (NJ) trees based on pairwise genetic distances. The quality of the branching patterns for NJ was assessed by bootstrap resampling of the data sets with 1,000 replications and rooted to Rhizopus oryzae CBS 112.07 T (NR 103595.1) and Trametes versicolor CFMR FP-135156-Sp T (NR 154494.1). The sequences determined in this study have been submitted to the ITS NCBI database and assigned Accession nos. MT889820 to MT889904.

Fungal tolerance to PAHs

The tolerance to hydrocarbons was tested by inoculating and cultivating the fungi in swMM supplemented with 25 mg/L of each fluoranthene, phenanthrene, pyrene and 5 mg/L of benzo[a]pyrene as only carbon source. The analytical grade PAHs (Aldrich Chemical Co) were added to the media as solution in acetone. Fungal strains were inoculated in the plates and incubated at 20 °C in darkness during 15 days in order to maintain culture condition closer to that observed in the environment. The capacity of fungi to grow and develop conidia was considered as tolerance while in absence of development the strain was classified as no-tolerant.

Fungal PAHs removal rates

Between 80-100 mg of mycelia and conidia were recovered of MDAsw plates and inoculated in 80 mL flasks with 30 mL of MDsw (1% MD). Fluoranthene, phenanthrene, pyrene and benzo[a]pyrene were then added from a stock solution prepared in acetone that contain 20 mg/L of each hydrocarbon. Samples were set for 1 hour before incubation to let acetone evaporate. An uninoculated flask was used as abiotic control and PAHs concentration reference. Cultures were incubated in darkness for 20 days at 20 °C with gentle shaking at 80 rpm, in order to maintain culture condition closer to that observed in the environment. Hydrocarbons were extracted after incubation adding 30 mL of ethyl acetate and shacked for 15 min at 600 rpm. The recovery yield was estimated to be about 98% of the initial concentration using the abiotic controls as reference.

Chrysene was used as internal standard during extraction in a concentration of 10 mg/L. Two milliliters of organic phase was pulled in a glass vial for its analysis in Gas Chromatography equipped with Flame Ionization Detector (GC-FID) (Agilent Technologies®, Network 6850 GC System) with a capillarity C18 reverse column (30 m*0.25 mm*0.25 µm). For the analysis, 1 µL was injected with a split ration of 1/50 using helium as carrier gas. Column temperature ramp settle from 200 to 240 °C with stepped temperature increase of 5 °C/min and held during 1 min at 240°C. Flame ionization detector was settled at 290 °C.

The removal capacities (degradation and/or sorption) for selected strains (Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76 strains exhibiting the highest and the lowest removal capacities, respectively) was determined in triplicate with an incubation period of 15 days at with gentle shaking at 80 rpm in order to maintain culture condition closer to that observed in the environment. PAHs extraction was performed as above described. Phanerochaete chrysosporium strain was used as reference for PAH-removal capacity, which often serves as reference for the comparison of PAH-removal capacities even between strains from different phyla, as it is the fungi the most studied in PAH-degradation [START_REF] Cao | Enzyme activities during Benzo[a]pyrene degradation by the fungus Lasiodiplodia theobromae isolated from a polluted soil[END_REF]. A one-way of analysis of variance (ANOVA) was used to assess the significance of PAH-removal differences between samples with a significance level of p < 0.05.

PCR detection of peroxidase and laccase genes

The presence of genes encoding for enzymes known to be related to PAHs degradation: laccase (lac), manganese peroxidase (MnP1, MnP2, MnP3) and lignin peroxidase (LiP1, LiP2, LiP3, LiP4, LiP5, LiP6) was checked by PCR amplification. Phanerochaete chrysosporium, an effective PAH degrader [START_REF] Bamforth | Bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions[END_REF][START_REF] May | Ex-Situ Process for Treating PAH-Contaminated Soil with Phanerochaete chrysosporium[END_REF], was used as positive control for the presence of the peroxidase genes. The sequences of the primers and the Tm for the amplification of the different genes are presented in Table 1. The reaction mix was prepared with 1 µL of extracted DNA in 9.5 µL of DEPC-treated water, 1 µL of each primer (20 µM), 12.5 µL AmpliTaq Gold 360 Master Mix 2X (Thermo Fisher Scientific, USA). The amplification was performed through 35 cycles of 95 °C (30 s), Tm (Table1, 45 s) and 72 °C (45 s), with a previous activation start of 95 °C (10 min) and final extension step at 72 °C (10 min). Peroxidase genes amplified fragments were sequenced at the Eurofins platform (France).

PAHs internalization and transport

The capacity to internalize and transport PAHs through hyphae was examined for selected strains (F. pseudonygamai F5.76 and A. destruens F10.81). The experimental setup consisted on an empty petri dish with two MDAsw cubes over a crystal slide with a separation of 6 mm between them.

One of the cubes contained pyrene at 30 mg/L while the other no. The fungi were inoculated in the cube with pyrene and incubated for 7 days in darkness at 20°C. The transport of PAHs was evaluated inside the mycelia that reach the cube without pyrene using an epifluorescence microscope (Nikon, Eclipse E600) with DAPI light filter (excitation 345 nm, emission 485 nm) for PAH detection (fluorescence wavelengths range from 210-380 nm) [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF].

Results and discussion

Identification of fungal strains Isolated from coastal sediments

In total, 85 fungal strains were isolated from PAHs contaminated coastal sediments in seawater media containing malt dextrose agar (swMDA). The strains were identified with the complete ITS sequence (including ITS1, 5.8S rRNA gene, and ITS2 regions), which provide accurate identification of fungi species even at the subspecies level [START_REF] Fajarningsih | Internal transcribed spacer (ITS) as DNA barcoding to identify fungal species: a review[END_REF]. The phylogenetic analysis showed that 83 strains belong to the Ascomycota Phylum and two strains belong to the Basidiomycota Phylum (Fig. 1 and2). Such result was not surprising since fungi belonging to Ascomycota have been found prevalent in marine sediments [START_REF] Babu | Fungi Diversity in Different Coastal Marine Ecosystem along South East Coast of India[END_REF][START_REF] Birolli | Biodegradation of anthracene and several PAHs by the marinederived fungus Cladosporium sp. CBMAI 1237[END_REF][START_REF] Ravelet | Biodegradation of pyrene by sediment fungi[END_REF] and other environments [START_REF] Reyes-César | Biodegradation of a mixture of PAHs by non-ligninolytic fungal strains isolated from crude oil-contaminated soil[END_REF]. The 85 fungal strains fall into six different Orders: Capnodiales (59 strains), Eurotiales (14 strains), Trichosphaeriales (1 strain), Hypocreales (2 strains), Pleosporales (7 strains) and Polyporales that belong to Basidiomicota Phylum (2 strains).

The isolated strains affiliated to Eurotiales included strains belonging to Talaromyces (T. helicus), Aspergillus (A. fumigatus and A. chevalieri), and Penicillium (P. glandicola, P. crustosum, and P. bialowiezense) genera (Fig. 1). Talaromyces and Aspergillus genera are known for their ability of PAH degradation in soil [START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF], while Aspergillus genera, especially A. fumigatus, has been detected in oil-contaminated mangrove sediments [START_REF] Ghizelini | Fungal communities in oil contaminated mangrove sediments -Who is in the mud?[END_REF]. The isolated strains related to the Nigrospora genus (Trichosphaeriales), N. rubi and N. gorlenkoana, are described for the first time in marine sediments. The presence of these strains in the sediments might be explained by plant material entering into the sea by air transportation or runoff, as they are known to be associated with plants [START_REF] Hao | Nigrospora Species Associated with Various Hosts from Shandong Peninsula, China[END_REF]. Similarly, the strains affiliated to the Hypocreales, Fusarium pseudonygamai (plant pathogen), Lecanicillium longisporum and Akanthomyces muscarius (entomopathogens) have been described only in soil so far [START_REF] Ansari | Evaluation of entomopathogenic fungi and a nematode against the soil-dwelling stages of the crane fly Tipula paludosa[END_REF][START_REF] Bashyal | Single and combined effects of three Fusarium species associated with rice seeds on the severity of Bakanae disease of rice[END_REF][START_REF] Danilovich | First report on the entomopathogenicity and virulence of Akanthomyces muscarius LY 72.14, a Yungas native fungal isolate, for Anastrepha fraterculus control[END_REF]. Regarding the Pleosporales, the strain F1.72, closely related to Neosulcatispora strelitziae and Phaeosphaeria podocarpi, recently described fungal species [START_REF] Crous | Fungal Planet description sheets[END_REF](Crous et al., , 2016)), represents probably also a novel fungal species. However, further analysis, including multi-locus based phylogeny, is required to characterize the strain. Two other strains were closely related to species within the Pleosporales, Alternaria destruens and Epicoccum poae, which have been isolated from plants [START_REF] Kumar | Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity[END_REF][START_REF] Chen | Didymellaceae revisited[END_REF]. So far, these strains have not been shown to exhibit hydrocarbon degradation capacity. The strains affiliated to the Polyporales were related to Trametes versicolor and Bjerkandera adusta that are known to be able to degrade hydrocarbon [START_REF] Lladó | A diversified approach to evaluate biostimulation and bioaugmentation strategies for heavy-oilcontaminated soil[END_REF][START_REF] Andriani | Effects of saline-alkaline stress on benzo[a]pyrene biotransformation and ligninolytic enzyme expression by Bjerkandera adusta SM46[END_REF].

All the Capnodiales were affiliated to two complexes of the Cladosporium genus (Fig. 2) defined by a multi-locus phylogeny [START_REF] Schubert | Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics[END_REF]. Among the Cladosporioides complex, the isolated strains were affiliated to species known to be associated with human and animals diseases such as Similarly, the isolated strains belonging to the Herbarum complex, C. rhusicola, C. subcinereum, C. angustiherbarum have been described involved in human and animals infections (Sandoval-Denis et al., 2016), while C. allicinum was found associated with marine organisms [START_REF] Poli | Fungal Diversity in the Neptune Forest: Comparison of the Mycobiota of Posidonia oceanica, Flabellia petiolata, and Padina pavonica[END_REF][START_REF] Bovio | Marine Fungi from the Sponge Grantia compressa: Biodiversity, Chemodiversity, and Biotechnological Potential[END_REF] and several strains related to C. herbarum have been described for their ability to degrade PAH in marine sediment [START_REF] Marco-Urrea | Potential of non-ligninolytic fungi in bioremediation of chlorinated and polycyclic aromatic hydrocarbons[END_REF][START_REF] Xiao | Stronger network connectivity with lower diversity of soil fungal community was presented in coastal marshes after sixteen years of freshwater restoration[END_REF]. Noteworthy, the strain D16.68 is the more distant from Cladosporium species (Fig. 2) suggesting that it might represent a novel species within the Cladosporium genus, but further phylogenetic analysis based on multi-locus are required to elucidate the taxonomic position. Although Cladosporium has been already reported in saline environments [START_REF] Zalar | Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments[END_REF], in hydrocarbon contaminated sediments [START_REF] Ravelet | Biodegradation of pyrene by sediment fungi[END_REF] showing as well resistance to metals [START_REF] Shao | Intracellular sequestration of manganese and phosphorus in a metal-resistant fungus Cladosporium cladosporioides from deep-sea sediment[END_REF], it was surprising to obtain mainly strains of this genus. It is likely that members of the Cladosporium genus are well adapted to the culture conditions imposed during the screening procedure. The cultural approach owns some limitations. Indeed, the development of conidia is controlled by different factors [START_REF] Tan | Substrate type and microbial interactions as factors affecting ascocarp formation by mangrove fungi[END_REF], such as the presence of PAHs [START_REF] Zafra | Morphological changes and growth of filamentous fungi in the presence of high concentrations of PAHs[END_REF], influencing the selection of cultivable strains. In order to overcome such limitations, the application of different culture conditions will enlarge the diversity of isolated strains.

Despite the limitations inherent of the cultivable approach, a large diversity of cultivable fungi was obtained from hydrocarbon-contaminated marine sediments, spanning 11 fungal genera. The isolated strains included not only strains affiliated to Orders which members were isolated from marine sediments [START_REF] Mouton | Diversity and Characterization of Culturable Fungi from Marine Sediment Collected from St. Helena Bay, South Africa[END_REF][START_REF] Ravelet | Biodegradation of pyrene by sediment fungi[END_REF] showing the capacity to degrade PAHs [START_REF] Fedorak | Oil-degrading capabilities of yeasts and fungi isolated from coastal marine environments[END_REF][START_REF] Simister | Degradation of oil by fungi isolated from Gulf of Mexico beaches[END_REF], but also some isolated strains yet not described in marine sediments, nor for their tolerance to the presence of PAHs. Thus, our study shows that a large fungal diversity remains hidden in marine sediments, which represent a metabolic potential for the development of remediation strategies for the mitigation of the effect of PAHs.

PAHs tolerance and removal capacities of the fungal isolated strains

Most of the isolated fungal strains (54 strains, 64%) were able to grow in the presence of at least the presence of one PAH showing their tolerance to hydrocarbons (Fig. 3). Among them, 61% tolerate the presence of benzo[a]pyrene, 52% pyrene and 45% fluoranthene. Few fungal strains were able to develop in presence of phenanthrene either alone (19%) or in mixture with other PAHs (14%). Similar results showing high tolerance of fungal strains to pyrene, and low tolerance to phenanthrene and PAHs mixture have been reported in the same range of concentrations [START_REF] Lee | Biotechnological procedures to select white rot fungi for the degradation of PAHs[END_REF]. Toxic effects on fungal growth have been observed with phenanthrene [START_REF] Lisowska | Microcalorimetry as a possible tool for phenanthrene toxicity evaluation to eukaryotic cells[END_REF] and metabolites produced from PAHs mixture [START_REF] Lundstedt | Degradation and formation of polycyclic aromatic compounds during bioslurry treatment of an aged gasworks soil[END_REF]. Interestingly, the tolerance capacity is consistent with the phylogeny (Fig. 3), the members of the same Order showing similar tolerance patterns. Noteworthy, the two groups Cladosporioides and Herbarum within the Capnodiales Order showed distinct tolerance capacities, which further support the classification into two distinct groups.

In order to assess the PAHs removal capacity (degradation and/or sorption) of fungal isolated strains, maltose and dextrose were added as extra carbon source, since fungi have been shown to have low ability to use PAHs as sole carbon source [START_REF] Harrison | Biodegradation of PAHs by fungi in contaminated-soil containing cadmium and nickel ions[END_REF]. In these conditions, fungal strains belonging to the Pleosporales Order showed the most efficient removal capacities while strains affiliated to the Hypocreales Order showed the lowest removal capacity (Fig. 3). In the Pleosporales Order, 85% of the strains showed removal capacity above 70%. The Cladosporoides group of the Capnodiales Order exhibited the less number of strains with removal capacities above 70%. The comparison of the removal capacities of the isolated strains showed two main clusters (Fig. 4) separating the strains with high removal capacities from those with low removal capacities.

Interestingly, members of the same species showed divergent removal capacities. Such discrepancies have been described [START_REF] Lee | Biotechnological procedures to select white rot fungi for the degradation of PAHs[END_REF], strains from the same species showing different metabolic capacities.

The analysis also showed that pyrene and fluoranthene (4 rings PAHs) clustered together, further confirmed by strong correlation between pyrene and fluorentene removal capacities (Pearson coefficient: 0.996, R 2 : 0.993), indicating that they were removed by almost a similar pattern of fungal strains (Fig. 3). The benzo[a]pyrene (5 rings PAH) and phenanthrene (3 rings PAH) were apart indicating that the patterns of fungal strains able to remove them were different. Such observations highlighted that the removal capacity depends also on the PAH structure as previously suggested [START_REF] Ghosal | Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A review[END_REF].

In order to further characterize the genetic PAH degradation potential of the isolated fungal strains, the presence of genes encoding manganese peroxidase (MnP), lignin peroxidase (LiP) and laccase (Lac), known to participate in the degradation of PAHs [START_REF] Ghosal | Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A review[END_REF], was examined in their genomes by PCR (Fig. 4). All strains posses at least one of these genes, the LiP2 being the most distributed (82/85 strains, 96%) among the isolated fungal strains (Fig. 4). Noteworthy, when the LiP2 gene was not present, the strain possessed the MnP2 gene. Almost all strains (80/85 strains, 94%) possessed at least a manganese peroxidase gene, MnP2 gene being the most detected (75/85 strains, 88%). Such results were not surprising since most of the peroxidase enzymes are known to be produced in marine environment [START_REF] Bonugli-Santos | Marine-derived fungi: diversity of enzymes and biotechnological applications[END_REF]. Surprisingly, the Lac gene, found in many marine fungal species [START_REF] Ali | Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications[END_REF][START_REF] Souza-Ticlo | A Thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus[END_REF], was detected in only 4 strains, which exhibited the most genetic potential possessing more than 5 of the targeted genes. However, since various types of Lac genes have been described in fungi [START_REF] Moreno | Phylogenomic analyses reveal the diversity of laccase-coding genes in Fonsecaea genomes[END_REF][START_REF] Yang | Laccase Production and Differential Transcription of laccase genes in Cerrena sp. in response to metal ions, aromatic compounds, and nutrients[END_REF], the primers used to detect the presence of Lac genes are probably not well suited for recovering the entire Lac gene diversity. The use of primers targeting broader Lac gene diversity or targeting at least Lac gene detected within the Ascomycota phyla (the major phyla of the isolated strains) is required to better define the presence of Lac genes in the isolated strains. Similar observations can be drawn for the LiP and MnP genes indicating that further efforts are needed for in depth characterization of the genetic potential of the isolated strains.

Interestingly, the genetic potential of Alternaria destruens F10.81, exhibiting the highest PAH removal capacity, was different to that of Fusarium pseudonygamai F5.76, showing the lowest removal capacity, by just the presence of the LiP1 gene. Although it cannot be excluded that the expression of the genes might be controlled by different regulation mechanisms in both strains, such observation suggested that the presence of the LiP, MnP and Lac genes was not linked with the PAH-removal capacity. In the same way, Pearson correlation analysis was unable to establish correlation between the genetic profiles (LiP, MnP and Lac) and PAH-removal capacity of the isolated fungal strains, but confirmation by determining the activity of the enzymes would be required in order to conclude on the involvement of the enzymes in PAH-removal. Anyway, considering the observed genetic potential and despite the inherent bias of the molecular tools used in our study, these results suggested that other mechanisms are probably involved in PAH removal. Thus, further studies are required for elucidating whether the PAH-removal potential of the isolated strains involves degradation and/or sorption mechanisms. The mechanisms described so far involve monooxygenase genes [START_REF] Cerniglia | Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation[END_REF][START_REF] Cerniglia | Degradation of Polycyclic Aromatic Hydrocarbons by Fungi[END_REF], particularly the intracellular P450 monooxygenase gene that implies the internalization of PAH into fungal cells [START_REF] Cerniglia | Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation[END_REF]. In order to determine whether the internalization of PAH and the hyphae PAH transport are mechanisms involved in PAH removal, the Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76 strains were selected, because they exhibited the highest and lowest PAH removal capacities respectively, for further characterization.

PAH removal characterization of Alternaria destruens F10.81 and Fusarium pseudonygamai

F5.76

The removal capacity of Alternaria destruens F10.81 and Fusarium pseudonygamai F576 was compared with that of Phanerochaete chrysosporium, which is the most studied fungi for PAHdegradation [START_REF] Cao | Enzyme activities during Benzo[a]pyrene degradation by the fungus Lasiodiplodia theobromae isolated from a polluted soil[END_REF]. It serve often as control fungi even for comparing PAH-removal capacity from strains belonging to different phyla [START_REF] Cao | Enzyme activities during Benzo[a]pyrene degradation by the fungus Lasiodiplodia theobromae isolated from a polluted soil[END_REF], because it exhibit the capacity to degrade a broad range of organic compounds [START_REF] Deschler | Involvement of 3,4-dichlorophenol hydroxylase in degradation of 3,4dichlorophenol by the white rot fungus Phanerochaete chrysosporium[END_REF][START_REF] Duran | Degradation of chlorophenols by Phanerochaete chrysosporium: effect of 3,4-dichlorophenol on extracellular peroxidase activities[END_REF], including several PAHs [START_REF] Pointing | Feasibility of bioremediation by white-rot fungi[END_REF]. Under our conditions P. chrysosporium presented low rates of PAHs removal (< 30%), just above to that exhibited by F. pseudoygamai F5.76 and around 3 times less to that observed for A. destruens F10.81 (Fig. 5). In fungi, gene regulation involves complex control mechanisms as those observed for peroxidases genes. It is known that in most fungal strains the LiP, MnP and Lac genes are expressed during the idiophase, the fungal secondary phase, when nitrogen is limited and under the control of complex regulation signals [START_REF] Junghanns | Degradation of the xenoestrogen nonylphenol by aquatic fungi and their laccases[END_REF]Kamitsuji et al., 2004;[START_REF] Knop | The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications[END_REF][START_REF] Duran | Degradation of chlorophenols by Phanerochaete chrysosporium: effect of 3,4-dichlorophenol on extracellular peroxidase activities[END_REF][START_REF] Solé | Differential regulation by organic compounds and heavy metals of multiple laccase genes in the aquatic hyphomycete Clavariopsis aquatica[END_REF], although the expression of MnP genes have been observed under high nitrogen content in fungal genera such as Pleurotus and Trametes [START_REF] Kaal | Increasing ligninolytic enzyme activities in several white-rot Basidiomycetes by nitrogen-sufficient media[END_REF][START_REF] Janusz | Fungal laccase, manganese peroxidase and lignin peroxidase: Gene expression and regulation[END_REF][START_REF] Stajić | Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species[END_REF]. Thus, the differences observed in removal capacities between the fungal strains are probably due to the medium composition and culture conditions.

It is likely that the seawater medium with high nitrogen content as well as the culture conditions used in our study limited the removal capacities of P. chrysosporium [START_REF] Singh | The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes[END_REF]) and F. pseudoygamai F5.76. In contrast, A. destruens F10.81 exhibited removal rates above 80% for all PAHs except for benzo[a]pyrene (65% removal; Fig. 5). Such higher PAH removal capacity of A. destruens F10.81 suggested that either its genes involved in PAH removal respond to different regulation signals than the other two strains or the PAH removal was performed by other mechanisms. For example, the expression of LiP, MnP and Lac genes has been observed under high nitrogen content in some fungal species (i.e. Pleurotus ostreatus and Trametes trogii) and even under both high and low nitrogen content for Dichomitus squalens, while for other fungal species, such as P. chrysosporium, the peroxidase genes are expressed under nitrogen limitation [START_REF] Janusz | Fungal laccase, manganese peroxidase and lignin peroxidase: Gene expression and regulation[END_REF][START_REF] Stajić | Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species[END_REF]. The expression of genes involved in PAH-removal even in high nitrogen content might be an asset for the fungal saprotrophic life-style in marine environments where secreted enzymes, such as peroxidases, are likely to be lost by rapid diffusion in the aquatic environment [START_REF] Richards | Marine fungi: their ecology and molecular diversity[END_REF].

Possible PAH removal has been described through biosorption mechanisms, which include adsorption onto cell surface [START_REF] Raghukumar | Removal of polycyclic aromatic hydrocarbons from aqueous media by the marine fungus NIOCC 312: involvement of lignin-degrading enzymes and exopolysaccharides[END_REF] and absorption into the cell [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF][START_REF] Yang | Removal of trace organic contaminants by nitrifying activated sludge and whole-cell and crude enzyme extract of Trametes versicolor[END_REF]. Several studies have demonstrated the capacity of fungi to uptake PAHs [START_REF] Deng | Cell surface properties of five polycyclic aromatic compound-degrading yeast strains[END_REF][START_REF] Wu | Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14[END_REF] and also to transport them along the fungal hyphae [START_REF] Furuno | Mycelia promote active transport and spatial dispersion of polycyclic aromatic hydrocarbons[END_REF][START_REF] Schamfuß | Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria[END_REF]. Both strains, Fusarium pseudoygamai F5.76 and Alternaria destruens F10.81, were able to uptake and transport pyrene (Fig. 6). Clear pyrene containing vacuoles were observed in F. pseudoygamai F5.76 (Fig. 6c,d) while pyrene was homogeneously distributed in A. destruens F10.81 (Fig. 6e,f) suggesting that the fungal strains have developed different strategies for PAH uptake. It has been demonstrated that the vacuoles serves for possible storage of PAHs as carbon source and for PAH transport along the hyphae allowing the distribution of PAH within the mycelia network [START_REF] Darrah | The Vacuole System Is a Significant Intracellular Pathway for Longitudinal Solute Transport in Basidiomycete Fungi[END_REF][START_REF] Furuno | Mycelia promote active transport and spatial dispersion of polycyclic aromatic hydrocarbons[END_REF]. Consistently, pyrene was also accumulated into conidia in F. pseudoygamai F5.76 (Fig. 6c,d), which represents carbon source reserve for the development of conidia as previously reported [START_REF] Allaway | Vacuolar Reticulum in Oomycete Hyphal Tips: An Additional Component of the Ca 2+ Regulatory System?[END_REF][START_REF] Bago | Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi[END_REF]. In contrast, the homogenous pyrene distribution in A. destruens F10.81 (Fig. 6e,f) suggested a diffusion mechanism. Such different pyrene uptake mechanism probably explains the highest removal capacities of A. destruens F10.81 in comparison to F. pseudoygamai F5.76 (Fig. 5).

However, further studies are required to determine whether higher pyrene absorption or internal degradation by monooxygenease (i.e. cytochrome P450) are the underlying physiological mechanisms of PAH removal in A. destruens F10.81.

Conclusion

The exploration of the cultivable fungal diversity of hydrocarbon-contaminated coastal sediments revealed that coastal sediment hide fungal diversity yet unexplored for their metabolic potential, especially regarding PAH removal capacity. A large proportion of the isolated strains (48%), dispatched within 6 fungal genera, exhibited PAH-tolerance with a removal capacity (degradation and/or sorption) above 60%. Such diversity in PAH-removal capacity represents a functional potential for ecosystem recovery exploitable for bioremediation treatments [START_REF] Harms | Untapped potential: exploiting fungi in bioremediation of hazardous chemicals[END_REF].

However, the mechanism underlying the PAH-removal capacity (degradation and/or sorption) is unclear because it is probably not related to the presence of extracellular peroxidase genes (LiP, MnP and Lac) and it is strain specific. The comparison of two isolated strains exhibiting contrasted removal capacities showed different PAH-uptake behaviour suggesting that the mechanisms by which fungi perform PAH-uptake might determine the efficiency of PAH-removal. Alternaria destruens F10.81, the most efficient PAH-remover (above 80%) was able to internalize pyrene homogenously into the hyphae that contrasted with the behaviour of Fusarium pseudoygamai F5.76 in which PAH-vacuoles were observed but exhibiting a PAH-removal capacity below 20%. It is likely that Alternaria destruens F10.81 owns features well adapted to PAH-contaminated coastal sediments, which represent potential for the development of a bioremediation process. However, further studies are required to understand the PAH-removal mechanism in order to manage fungal resources to mitigate the effects of PAH contamination.

Acknowledgment

We acknowledge the support of the French/German bilateral program PHC-PROCOPE FungDeg (Elucidating biomarkers and functions of fungal bacterial interactions during the degradation of polycyclic aromatic hydrocarbons at changing environmental conditions; project no. 42512WL).

We would like to thank all partners of the FungDeg project and MELODY group for their useful discussions. JAB received a fellowship from CONACyT number 326250/471689 and a mobility grant from the centre of excellence E2S at Pau University.

Figure captions

C

  . crousii, C. welwitschiicola, C. austroafricanum, C. pini-ponderosae, and C. puyae (Sandoval-Denis et al., 2016), and with marine organisms such as C. colombiae[START_REF] Ravi Theja | Molecular characterization of fungi isolated from sea urchin Stomopneustes variolaris (Lamarck, 1816) -st. mary's island, west coast of india[END_REF].

Fig. 1 :

 1 Fig. 1: Phylogenetic tree, based on ITS sequences, showing the positions of fungal strains isolated from oil contaminated coastal marine sediments. The Neighbour joining phylogenetic tree was rooted with the ITS sequence of Rhizopus orzyzae CBS 112.07 T (NR 103595.1). The scale bar corresponds to 0.05 substitutions per nucleotide position. Percentages of 1,000 bootstrap resampling that supported the branching orders in each analysis are shown above or near the relevant nodes. For the isolates, the number of isolated strains is indicated in parenthesis. For the type strains, the accession number is indicated in parenthesis.

Fig. 2 :

 2 Fig. 2: Phylogenetic tree, based on ITS sequences, showing the positions of fungal strains isolated from oil contaminated coastal marine sediments within the Cladosporium genus. The Neighbour joining phylogenetic tree was rooted with the ITS sequence of Trametes versicolor CFMR FP-135156-Sp T (NR 154494.1). The scale bar corresponds to 0.05 substitutions per nucleotide position. Percentages of 1,000 bootstrap re-sampling that supported the branching orders in each analysis are shown above or near the relevant nodes. For the isolates, the number of isolated strains is indicated in parenthesis. For the type strains, the accession number is indicated in parenthesis.

Fig. 3 :

 3 Fig. 3: PAHs tolerance and removal capacities of isolated fungal strains. PAHs tolerance corresponds to the capacity of the fungal strains to grow (green) or not (red) on solid seawater minimal medium in the presence of different PAHs and PAHs mixture. PAHs removal capacity, determined in liquid cultures containing a mixture of PAHs, corresponds to the percentage of PAHs eliminated after 20 days of fungal growth. The color gradient follows to the removal capacity from low (red) to high (green). Phe, phenanthrene; Flu, fluoranthene; Pyr, pyrene; BaP, benzo[a]pyrene; Mix, mixture of the 4 PAHs.

Fig. 4 :

 4 Fig. 4: Comparison of PAHs removal capacity of the isolated fungal strains and their genetic potential. The heatmap is based on similarity index calculated from removal capacity data shown in Fig 3. Phe, phenanthrene; Flu, fluoranthene; Pyr, pyrene; BaP, benzo[a]pyrene. The genetic potential corresponds to the presence (dark blue) of peroxidase genes assessed by PCR targetingmanganese peroxidase (MnP1-3), lignin peroxidase (LiP1-6) and laccase (Lac). The absence of the genes is indicated in pale blue. The fungal strains selected for further analysis, corresponding to the highest and lowest removal capacities, are highlighted in red.

  Fig. 1

  Fig. 2