Oumayma Kahla

Sondes Melliti

Ben Garali

Manel Fatma Karray

Manel Ben Abdallah

Najwa Kallel

Najla Mhiri

Hatem Zaghden

Badreddine Barhoumi

Olivier Pringault

Marianne Quemeneur

Badreddine Bahoumi

Marianne Quémeneur

Marc Tedetti

Sami Sayadi

Asma Sakka Hlaili

Efficiency of benthic diatom-associated bacteria in the removal of benzo(a)pyrene and fluoranthene

Keywords: Accumulation, biodegradation, Bizerte Lagoon, Nitzschia sp. phycosphere, hydrocarbondegrading bacteria

Introduction

Marine pollution through chemical contaminants has become a matter of great concern because of its deleterious impacts on marine ecosystems. Polycyclic aromatic hydrocarbons (PAHs) are among the most hazardous pollutants, with potential harmful effects on biota and ecosystems [START_REF] Hylland | Polycyclic aromatic hydrocarbon (PAH) ecotoxicology in marine ecosystems[END_REF]. They are hydrophobic and ubiquitous pollutants detected in all ecosystems, from polar regions to the tropics [START_REF] Wilcke | Global patterns of polycyclic aromatic hydrocarbons (PAHs) in soil[END_REF][START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF].

Sixteen PAHs have been included in the US Environmental Protection Agency's list of priority pollutants (US-EPA). Because of their strong toxicity, persistence, and accumulation in marine animals and plants, a great deal of attention has been paid to the elimination and degradation of 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] González-Gaya | Biodegradation as an important sink of aromatic hydrocarbons in the oceans[END_REF]. Physicochemical and thermal techniques are used in several processes of soil or sediment treatments, but biological remediation has been recognized as the most efficient method for decontaminating environments polluted by PAHs. Indeed, PAHs are biodegraded by a wide range of microorganisms [START_REF] Mckew | Determining the identity and roles of oil-metabolizing marine bacteria from the Thames Estuary, UK[END_REF][START_REF] Gutierrez | Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP[END_REF][START_REF] Thompson | Enhanced crude oil biodegradation potential of natural phytoplankton-associated hydro-carbonoclastic bacteria[END_REF]. Among them, bacteria are considered as the dominant agents in hydrocarbon biodegradation, and several marine hydrocarbon-degrading isolates have been described worldwide [START_REF] Ben Said | Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia[END_REF][START_REF] Haritash | Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review[END_REF][START_REF] Jiménez | Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential[END_REF]. Thus, "marine bacterial consortia" have been widely used in efforts to depollute environments contaminated by PAH mixtures and oil, such as harbors and wastewater effluents, or during oil spills [START_REF] Arulazhagan | Role of a moderately halophilic bacterial consortium in the biodegradation of polyaromatic hydrocarbons[END_REF]Vasudevan, 2009, Jiménez et al., 2011). Microalgae can also play a significant role in the removal of PAHs. Warshaskyet al. (1988) have shown that the freshwater microalgae Selenastrum capricornitum was able to metabolize benzo(a)pyrene (BaP) to cis-dihydrodiols through a system of enzyme dioxygenases such as that found in bacteria.

Two diatom species, Nitzschia sp. and Skeletonema costatum, can accumulate and degrade phenanthrene (Phe) and fluoranthene (Flt) [START_REF] Hong | Accumulation and biodegradation of phenanthrene and fluoranthene by the algae enriched from a mangrove aquatic ecosystem[END_REF]. Diatoms of the genus Pseudonitzschia have recently been reported to accumulate and degrade a mixture of PAHs (Melliti Ben Garali, 2016). Other studies have reported that Phe and pyrene (Pyr) could be converted by photosynthetic microorganisms into soluble diols, phenols, lactones, naphthoic acid, and phthalic acid, which may be excreted in the overlying water column [START_REF] Seo | Degradation of phenanthrene by Burkholderia sp. C3: initial 1, 2-and 3, 4-dioxygenation and meta-and ortho-cleavage of naphthalene-1, 2-diol[END_REF].

Other works have demonstrated that a "microalgae-bacteria consortium" could be successfully used in the treatment of aromatic pollutants ( [START_REF] Borde | Synergistic relationships in algal-bacterial microcosms for the treatment of aromatic pollutants[END_REF][START_REF] Muñoz | Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors[END_REF].

There is a synergetic relationship between algae and bacteria, in which algae provide oxygen (through photosynthesis) required by aerobic bacteria in the aerobic biodegradation of PAHs. In turn, bacteria produce carbon dioxide for photosynthetic metabolism. [START_REF] Borde | Synergistic relationships in algal-bacterial microcosms for the treatment of aromatic pollutants[END_REF] have shown that the removal of Flt and Pyr was better (up to 85%) when the green microalga Chlorella sorokiniana was cultivated with bacteria. Other studies have also reported that the use of microalgae is an efficient bioremediation strategy for PAH removal, since phototrophs can also co-metabolize PAHs together with bacteria [START_REF] Haritash | Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review[END_REF]. Indigenous microalgaebacteria consortia have effectively been applied in the biodegradation of crude oil and naphthenic acids water [START_REF] Tang | Construction of an artificial microalgal-bacterial consortium that efficiently degrades crude oil[END_REF][START_REF] Mahdavi | In situ biodegradation of naphthenic acids in oil sands tailings pond water using indigenous algae-bacteria consortium[END_REF]. More recently, studies have shown that bacteria associated with microalgae, the phycosphere, could play a significant role in hydrocarbon degradation [START_REF] Thompson | Enhanced crude oil biodegradation potential of natural phytoplankton-associated hydro-carbonoclastic bacteria[END_REF]2018). Hence, the phycosphere has attracted considerable interest regarding its potential in pollutant degradation, and scientists have focused on its structure, ecology and interaction with algae. Studies have shown that the bacteria associated with algae, which comprise both algae-attached and free-living bacteria, are specific to algal species, as algae can release strain-dependent organic compounds that are used by specific bacteria [START_REF] Bagatini | Host-specificity and dynamics in bacterial communities associated with bloom-forming freshwater phytoplankton[END_REF]. The presence of oil-and hydrocarbon-degrading bacteria within the phycosphere has been reported for dinoflagellates and diatoms [START_REF] Gutierrez | Algiphilus aromaticivorans gen. nov., sp. nov., an aromatic hydrocarbon-degrading bacterium isolated from a culture of the marine dinoflagellate Lingulodinium polyedrum, and proposal of Algiphilaceae fam[END_REF][START_REF] Mishamandani | Response of the bacterial community associated with a cosmopolitan marine diatom to crude oil shows a preference for the biodegradation of aromatic hydrocarbons[END_REF][START_REF] Severin | Dynamics of Heterocapsa sp. and the associated attached and free-living bacteria under the influence of dispersed and undispersed crude oil[END_REF][START_REF] Thompson | Enhanced crude oil biodegradation potential of natural phytoplankton-associated hydro-carbonoclastic bacteria[END_REF][START_REF] Thompson | Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH[END_REF]. These associated bacteria can influence the phytoplankton taxon-specific response to hydrocarbon pollution and oil spills [START_REF] Severin | The phytoplankton taxon-dependent oil response and its microbiome: correlation but not causation[END_REF]. Most studies have concerned planktonic microalgae and their associated bacteria, but little is known about the PAH biodegradation potential of microbenthic organisms. Benthic diatoms, thriving in contaminated sediments, can acquire physiological abilities to tolerate and metabolize pollutants [START_REF] Kottuparambil | PAHs sensitivity of picophytoplankton populations in the Red Sea[END_REF].

Considering these findings, the present study assessed the role of an indigenous benthic diatom and associated bacteria (co-culture, non-axenic) or benthic diatom alone (axenic culture) in the removal of two PAHs of interest, i.e. BaP and Flt. These two PAHs differ in structure/molecular weight and hydrophobicity/water solubility, leading to potentially different degradation capacities by microalgae and bacteria. Subsequently, the structures of bacteria associated with the diatom (attached or free-living) were also evaluated by Illumina MiSeq 16S rRNA sequencing analysis. Finally, isolated strains from associated bacteria were screened for their potential use of crude oil for growth. The diatom was isolated from a sediment of the Bizerte Lagoon (Tunisia), where high levels of PAHs have been recorded, with Flt and BaP as dominant compounds (265 and 168 μg kg -1 dry wt., respectively) [START_REF] Lafabrie | Contaminated sediment resuspension induces shifts in the structure and the functioning of phytoplankton in an eutrophic Mediterranean lagoon[END_REF][START_REF] Pringault | Consequences of contaminant mixture on the dynamics and functional diversity of bacterioplankton in a southwestern Mediterranean coastal ecosystem[END_REF]. Furthermore, previous works have highlighted that the indigenous microorganisms of the lagoon (bacteria or diatoms) can potentially biodegrade PAHs [START_REF] Ben Said | Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia[END_REF]Melliti Ben Garali, 2016). Therefore, it seems relevant to evaluate their co-metabolic synergy for a more efficient and faster bioremediation.

Material and methods

Microalgae isolation, culture, and identification

An indigenous diatom was isolated from sediment of the Bizerte Lagoon (north of Tunisia) and then cultured according to the protocol described by [START_REF] Lundholm | Buried alive -germination of up to a century-old marine protist resting stages[END_REF] and slightly modified by Melliti Ben Garali et al. (2016). Sediments were collected using a Van Veen grab (Hydrobios) at a station (37°15'40.22''N and 9°51'30.49''E) located in front of a cement manufactory, which is highly contaminated by PAHs [START_REF] Lafabrie | Contaminated sediment resuspension induces shifts in the structure and the functioning of phytoplankton in an eutrophic Mediterranean lagoon[END_REF][START_REF] Barhoumi | Polycyclic aromatic hydrocarbons (PAHs) in surface sediments from the Bizerte lagoon, Tunisia: levels, sources and toxicological significance[END_REF].

The sediment was sieved through a 2-mm mesh to remove coarse debris. In the laboratory, 3 g of fresh sediment were mixed with 90 mL of a sterilized f/2 hydroponic culture medium [START_REF] Guillard | Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve[END_REF] and incubated for 6 days in a thermostatic chamber at 22 o C, illuminated with cool-white fluorescent tubes at a light intensity of 100 µmole photons m -2 s -1 and under a photoperiod of 12 light: 12 dark. During incubation, the mixture was gently stirred to allow the release of microalgae from the sediments and their recovery to the surface. The supernatant, containing the cells, was taken and served to isolate the dominant diatom. Samples with live cells were examined under an inverted microscope (CETI, Versus, Belgium), and single cells were isolated using a glass Pasteur pipette into a tissue culture plate containing f/2 medium. All cultures were kept under the conditions described previously. The strain was re-inoculated into fresh medium at 2-week intervals. Cells from the culture were observed under a light microscope BX-102. The diatom was identified as a species of Nitzschia, based on the morphometric characteristic described by Bouchouicha Smida et al. ( 2014) (width, length, and form of the valves; number and form of the chloroplasts; presence or absence of the central interspace).

Sampling of attached and free-living bacteria

Tbtain attached bacteria (AB), 200 mL of the Nitzschia sp. culture were filtered through a 5.0-µm Nuclepore polycarbonate membrane. The free-living bacteria (FB) were recovered by collecting 5.0 µm of the filtrate onto a 0.22-µm cellulose polyester filter. These filters (two filters of 0.5 µm and two filters of 0.22 µm) were used for microbiological and molecular experiments.

DNA extraction, qPCR quantification, and Illumina Miseq sequencing and analysis

Total DNA was extracted from AB and FB filters (0.5 and 0.22 µm, respectively) using the Ultra Clean Water DNA kit (MO BIO), following the instructions of the manufacturer. The quantification of the DNA obtained and assessment of its purity were performed using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).

The abundance of total bacteria in AB and FB DNAs was estimated by real-time PCR targeting 16S rRNA genes, using the primer sets 331F/797R. The qPCR was made in triplicate in a Bio-Rad CFX-96 real-time system (Bio-Rad). The reaction components and qPCR protocol conditions have been described previously [START_REF] Ben Abdallah | Abundance and diversity of prokaryotes in ephemeral hypersaline lake Chott El Jerid using Illumina Miseq sequencing, DGGE and qPCR assays[END_REF]. The abundance of total bacteria was reported as DNA copy numbers of the corresponding gene per mL, using the standard curves.

The AB and FB DNAs samples were subjected to sequencing on an Illumina Miseq platform (CBS, SFAX, TUNISIA), using a paired-end 300-bp sequence read running with the Miseq Reagent Kit V3 (600cycles). The V3-V4 regions of prokaryotic 16S rRNA genes were PCRamplified with primers Pro341/Pro805R [START_REF] Takahashi | Development of a prokaryotic universal primer for simultaneous analysis of bacteria and archaea using next-generation sequencing[END_REF], using KAPA HiFi HotStart ReadyMix (2X) (KAPA Biosystems, Kit Code KK2602). The DNA samples were independently amplified in triplicated 25 μL reactions containing 0.1-10 ng target DNA, 1× Taq PCR Master Mix, and 400 nM of each primer. The PCR program was 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C for 30 cycles, followed by 10 min of final primer extension. The products of PCR products were purified using AMPure XP beads (Beckman Coulter, USA) following the manufacturer's recommendations and analyzed with a BioAnalyzer DNA 1000 Chip Kit (Agilent Technologies) and the Qubit ® ds DNA HS Assay Kit (Life Technologies). Taxonomic analyses of sequence reads were performed with the QIIME version 1.9.1 software package [START_REF] Caporaso | QIIME allows analysis of highthroughput community sequencing data[END_REF].

Chimera detection was performed with UCHIME, and chimeric sequences were filtered and discarded prior to further analysis [START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF]. Operational taxonomic units (OTUs) were assigned at 97% similarity threshold with the UCLUST algorithm [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF]. Taxonomic assignments were performed with the Greengenes 13.8 database. Sequences from selected dominant OTUs (> 1% of all sequences) were compared with related sequences retrieved from NCBI databases using BLAST. The 16S rRNA gene sequences determined in this study were deposited in the GenBank database under accession numbers MT229144 to MT229165.

For AB and FB communities, alpha diversity indices including Chao1 richness estimator, observed species and diversity indices [START_REF] Shannon | The mathematical theory of communication[END_REF][START_REF] Simpson | Measurement of diversity[END_REF], as well as the phylogenetic diversity (PD whole-tree) index [START_REF] Lozupone | Species divergence and the measurement of microbial diversity[END_REF]

Isolation, identification, and screening for bacteria growing on crude oil

Isolation

In an attempt to isolate strains of attached and free-living bacteria, the filters (5.0 and 0.22 µm, respectively) were transferred to sterile tubes containing physiological water (NaCl 9‰).

Aliquots (100 mL) of serial dilutions were plated onto solid Luria-Bertani as rich medium, containing (per liter): 10 g (w/v) tryptone (Bio Basic, Canada); 5 g (w/v) yeast extract (Panreac, Espagne); 5 g (w/v) NaCl (Bio Basic, Canada), and 20 g (w/v) agar (Bio Basic, Canada). The pH was adjusted to 7 with 10 M KOH before autoclaving. After 1-3 days of incubation at room temperature (approximately 27°C), yellowish, cream, and white colonies were obtained. Different colonies were picked and re-streaked several times to obtain pure cultures and then stored at -80°C in the isolation medium supplemented with 30% glycerol. Cell morphology was observed at 100

x magnification under oil immersion (Nikon Optiphot, Tokyo, Japan).

Molecular identification

Genomic DNA of both FB and AB isolates was extracted using a GF1-vivantis Nucleic acid extraction kit according to the manufacturer's protocol. The 16S rRNA genes were amplified using primer sets FD1 (5'-AGAGTTTGATCCTGGCTCAG-3') [START_REF] Weisburg | 16S ribosomal DNA amplification for phylogenetic study[END_REF]/1492R (5'-GGTTACCTTGTTACGACTT-3') [START_REF] Lane | 16S/23S rRNA sequencing[END_REF]. The PCR cycling conditions were the same as previously described [START_REF] Karray | Extracellular hydrolytic enzymes produced by halophilic bacteria and archaea isolated from hypersaline lake[END_REF]. The PCR products were sequenced using the Big Dye ®

Terminator cycle Sequencing kit and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) with FD1 and 1492r primers. Phylogenetic analysis of 16S rRNA gene sequences was performed as previously described [START_REF] Ben Abdallah | Abundance and diversity of prokaryotes in ephemeral hypersaline lake Chott El Jerid using Illumina Miseq sequencing, DGGE and qPCR assays[END_REF]. The 16S rRNA gene sequences of the bacterial isolates were deposited in GenBank under accession numbers MN517112 to MN517117.

Screening for bacteria growing on crude oil

The isolated strains were grown aerobically at room temperature in mineral salt medium (MSM) containing (per liter): (NH4)2SO4, 1g; NaH2PO4, 0.8; K2HPO4, 0.2; MgSO4, 0.2; CaCl2, 0.1; FeCl3, 0.005; MnSO4, 0.0002; ZnSO4, 0.0001; CuSO4, 0.00002; yeast extract, 0.5 g (pH = 7) (Yirui et al., modified 2009). Cells were cultured in 250-mL flasks containing 100 mL of liquid medium with 1 g of crude oil (BAL 150 type Arabian light) for approximately 1 month. A control (without inoculation) was included. Strain evolution was followed by microscopic observation, and their capacity to grow in the presence of crude oil was detected by visual changes in the culture (i.e., clear appearance indicated use of crude oil).

PAHs contamination experiment

Chemicals and preparation of PAH solution

Two concentrated solutions of BaP and Flt were prepared with HPLC-grade dimethylsulfoxide (DMSO, Sigma Aldrich D4540), and both PAH solutions were kept in the dark at 4°C prior to use. The PAHs (purity > 98%) were purchased from Sigma-Aldrich. Stock solutions of BaP and Flt (0.12 and 2.4 g L -1 , respectively) were prepared by dissolving pure chemicals in reagent-grade dimethylsulfoxide (DMSO, 0.05%) without exceeding the solubility threshold.

Experimental set up

The contamination experiment was conducted under the following conditions: non-axenic, axenic (without bacteria), and sterile (without microorganisms). For the non-axenic condition, nine

Erlenmeyer flasks (2 L, Schott-Duran glass), containing 150 mL of exponential diatom culture (initial concentration of 1.1 10 4 cells mL -1 ) and fresh f/2 medium, were used to carry out three treatments in triplicate: control treatment without any PAH (treatment C); treatment contaminated with Flt and treatment contaminated with BaP. The Flt and BaP were added to achieve final concentrations of 265 and 3 µg L -1 , respectively. These levels were very close to their respective solubility. For the axenic condition, the culture medium was autoclaved and sterilized with a mixture of two antibiotics (10,000 penicillin units and 10 mg of streptomycin per mL, Sigma Aldrich, PO781,) according to the protocol of [START_REF] Réveillon | Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum, Chaetoceros sp., Chaetocero scalcitrans and Thalassiosira pseudonana) and bacteria isolated from a diatom culture[END_REF]. Then, the three treatments (C, Flt, and BaP) were subjected to the same conditions as the non-axenic treatment. The axenic and non-axenic treatments (C, Flt, and BaP) were incubated for 7 days in a thermostatic chamber under the same conditions used for diatom cultures. The light intensity and the temperature within the incubator were checked throughout the experiment, using a spherical quantum mini-recorder LI-250 A and a thermometer (Model TH-020), respectively. Sterile treatments were performed in triplicates, in which the Erlenmeyer flask contained only f/2 culture medium (for treatment C) and f/2 medium with the PAHs for the treatments Flt and BaP. The sterile treatments were incubated for 7 days in light or in dark and settled to determine PAH removal by abiotic processes, i.e., photooxidation, volatilization, and adsorption on glass walls (see below).

PAH analysis

The PAHs were analyzed at the beginning and the end of the incubation in axenic and nonaxenic cultures as well as in sterile Erlenmeyer flasks in the light and the dark. Sub-samples (100 mL) were filtered through precombusted Whatman GF/F filters, and the filtrate and suspended particulate matter (SPM) adsorbed to the filters were used to extract dissolved and particulate PAHs, respectively.

Extraction of the SPM-loaded filters was conducted as previously described [START_REF] Liu | Spatial-temporal distributions, sources of polycyclic aromatichydrocarbons (PAHs) in surface water and suspended particular matter from the upper reach of Huaihe River, China[END_REF], with some modifications. Briefly, after freeze drying and weighing, the filters were spiked with surrogate standard (1-methylpyrene) and ultrasonicated for 1 h at 30°C with 30 mL of a mixture of n-hexane:acetone (1:1, v/v). Subsequently, the obtained extract was concentrated to about 5 mL in a rotary evaporator. Additional 10 mL of n-hexane were added to the pear-shaped flask and evaporated down to few hundreds of µL. This concentrated extract was purified through a glass column packed with 1 g anhydrous sodium sulfate in the upper part and 2 g of silica gel (previously activated by heating at 150°C overnight before use) in the lower part. After conditioning with 8 mL of n-hexane, the fraction containing PAHs (Flt and BaP) was eluted with 10 mL of the mixture of n-hexane and dichloromethane (1:1, v/v). The eluents were concentrated to 1-2 mL, subjected to solvent exchange to acetonitrile, and concentrated to 1.0 mL by a rotary evaporator prior to HPLC analysis. Extraction of dissolved PAHs was performed by Liquid-Liquid extraction (LLE) with 15 mL of dichloromethane (DCM) for 5 min. The extraction was repeated three times, and the obtained extracts were combined. The subsequent steps were identical as those described for SPM-loaded filter pretreatment.

Quantification of Flt and BaP was conducted using an analytical HPLC unit (JASCO, Japan)

equipped with a JASCO PU-2089 HPLC pump, a type 7125 Rheodyne injector (with a 20 μL loop), and a fluorescence detector (FP-2020) with excitation and emission wavelengths that could be varied throughout the analysis (Ex/Em: 288/462 nm (Flt), Ex/Em: 290/430 nm (BaP).

Separation was carried out using a SUPELCOSIL LC-PAH (Supelco, Inc. Bellefonte, PA) reversephase C18 column (4.6×250 mm, 5 μm particle size) specific for PAH analysis. Acetonitrile was used as mobile phase (isocratic elution mode), operated at a flow rate of 1 mL min -1 ; injection volume was 20 μL. We identified Flt and BaP via comparison of their retention time with those of the authentic standards and quantified them using the internal calibration method.

All data were subjected to strict quality control procedures. A series of solvent blanks (to determine any background contamination), spiked blanks, and a spiked matrix (to monitor recovery efficiency) were analyzed during the treatment and analysis procedures. Solvent blanks showed that Flt and BaP were detected in small amounts (< 5% of sample values). The spiked recoveries of Flt and BaP in water and filters were set in the range 82.3-100.6% and 95.7-113.3%.

Method detection limits (MDLs), defined as mean blank value + 3 × standard deviation (SD), ranged from 0.02 to 0.10 ng mL -1 in water and filters. The concentrations of Flt and BaP were blank corrected, but not corrected with recoveries. All solvents used for sample processing and analyses (DCM, hexane, acetone, and acetonitrile) were of high-performance liquid chromatography (HPLC) grade and were purchased from Fisher (UK). All glassware was intensively cleaned before using.

Chl a analyies

Subsamples (50 mL) from axenic and non-axenic cultures were taken daily and filtered through glass microfiber filters (GF/F, Whatman). The Chl a was extracted with 90% acetone (v/v) for 30 h in the dark at 5 o C. The pigment concentration was measured using the spectrophotometric method provided by [START_REF] Lorenzen | Determination of chlorophyll in sea water[END_REF], following the procedure described in [START_REF] Parsons | A manuel of Chemical and Biological Methods for Seawater analysis[END_REF].

Growth kinetics

To determine the diatom growth kinetics, subsamples (1 mL), taken daily from replicates of axenic and non-axenic treatments, were fixed with Lugol's acid solution (3% final concentration).

Cell counting was performed in triplicate on aliquots of 5 μl, deposited between slide-coverslip and observed under a light microscope BX-102 A at × 40 magnification [START_REF] Lundholm | Effect of pH on growth and domoic acid productionby potentially toxic diatoms of the genera Pseudo-nitzschia and Nitzschia[END_REF].

The exponential growth rate was calculated by considering three successive counts made in the exponential growth phase, according to the formula Nt = N0 e μt and ln (Nt / N0) = µ t, where N0

and Nt are the initial and final abundance values of the diatom, respectively; μ (d -1 ) is the net cell growth rate; t (d) is the incubation time; µ is the slope of the line from the linear regression of ln (Nt / N0) against time of the form y = a x.

Bacterial monitoring in axenic cultures

To ensure the success of the axenization method, total bacteria were enumerated via realtime PCR targeting 16S rRNA genes, using the primer sets 331F/797R as described above (Paragraph 2.3). Subsamples were collected from axenic cultures at the beginning of the experiments and after 3 and 7 days. Subsamples (200 mL) were filtered (0.5 and 0.22 µm), and total DNA was extracted from filters as described in Paragraph 2.3.

Determination of the relative contribution of the different PAH removal processes

The percentages of dissolved PAHs (i.e., in the medium, %PAHdis), accumulated in the cells (%PAHacc), and degraded (%PAHdeg) were calculated as follows [START_REF] Chan | Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum[END_REF]: in the light: %PAHdeg = %PAHvol + %PAHads + %PAHpho (Eq. 2)

%PAHdis=
So, Eq2 -Eq1 = %PAHpho

The %PAHvol + %PAHads and %PAHpho were considered as equivalent for all treatments and conditions.

Axenic condition

%PAHdeg= %PAHvol + %PAHads + %PAHpho + %PAHbiodeg (Eq. 3)

Therefore, Eq3 -Eq2 = %PAHbiodeg

In axenic cultures, biodegradation was due to the diatom, and therefore, the %PAHbiodeg was equal to the %PAHdiadeg, which would not differ considerably from that of the non-axenic culture.

Non-axenic condition

%PAHdeg= %PAHvol + %PAHads + %PAHpho + %PAHbiodeg (Eq. 4)

Therefore, Eq4 -Eq2 = %PAHbiodeg

In non-axenic cultures, biodegradation was due to both diatom biodegradation and bacterial biodegradation; therefore, the %PAHbiodeg minus the %PAHdiadeg equaled to %PAHbacdeg.

Statistical analysis

Statistical analyses were performed using the SPSS software version 14.0 for Windows.

Analysis of variance (ANOVA) was used to test the effects of BaP and Flt on Chl a, cell density, and growth rate. When the effect was significant, a multiple comparison posterior test (Tukey´s test) was performed to compare treatments in a 2 x 2 design. When the normality of data distribution (test of Kolmogorov-Smirnov) and/or the homogeneity of the variances (Bartlett-Box test) could not be verified, a non-parametric ANOVA (Kuskal-Wallis) was used. A student test was performed to compare the fractions of PAH remaining, accumulated, and degraded between axenic and non-axenic conditions and the fraction of PAHs degraded by diatoms and bacteria.

Results

Responses of Nitzschia sp. to BaP and Flt

The diatom maintained in the non-axenic control showed a pronounced growth, as the Chl a concentration increased significantly from an initial value of 37 ± 2.6 to 177 ± 19.5 μg L -1 at the end of the experiment (Fig. 1a). In BaP and Flt treatments, Chl a also showed an increase throughout the incubation, with slightly lower concentrations at the end of the experimental period (134 -137 ± 10.5 -19.5 μg L -1 ) relative to the control (Fig. 1a). In non-axenic cultures, the overall effect of the two PAHs on biomass was not significant (P > 0.05). In the control, Nitzschia sp.

exhibited a growth kinetics characterized by a 2-day latency, followed by exponential growth until the end of the incubation period, where it reached extremely high densities (31 ± 5.2 10 4 cells mL - 1 ) (Fig. 1b). In both PAH treatments, the diatom showed the same pattern of proliferation; growth rates (0.44 ± 0.009 d -1 and 0.45 ± 0.0003 d -1 ) were not significantly different from those of the control (0.50 ± 0.01 d -1 ) (P > 0.05, Table 1).

The axenization of the diatom was successfully maintained during the experiment, as no bacterial abundance was detected by qPCR throughout the incubation period. The axenic strain of Nitzschia sp. showed continuous proliferation in the control, with Chl a concentrations (18-92 μg L -1 ) and cell density levels (0.75-17 10 4 cells mL -1 ) significantly lower than those in the non-axenic control (37-177 μg Chl a L -1 ; 1-31 10 4 cells mL -1 ), concomitant with the lower growth rate (Table 1). Contamination with Flt caused a slight decrease in cell density (Fig. 2d), but the growth rate remained similar (0.41 ± 0.016 d -1 ) to that of the control (0.43 ± 0.03 d -1 ) (Table 1). In contrast, BaP addition provoked a significant decrease in Chl a and cell density (Fig. 1c,d), together with a decrease in growth rate (0.35 ± 0.002 d -1 ) relative to the control (Table 1). When comparing the responses of the axenic and non-axenic diatom strains, the most pronounced decrease induced by PAHs (especially for BaP) on Nitzschia growth was observed in the axenic culture.

Fate of BaP and Flt

The concentrations of Flt and BaP, measured in each treatment at the beginning of the experiment, were compared with those used theoretically for contamination (Table 2). The results

show that the measured levels were close to 95% of the theoretical concentrations. Therefore, the measured concentrations were used in the remainder of the results.

For all treatments and conditions, PAH levels remaining at the end of the experiment were low (maximum 8.5 ± 1.4% for BaP). In contrast, PAH degradation (%PAHdeg) and PAH accumulation in the cells (%PAHacc) were the dominant processes involved in PAH removal (Table 3). However, the %PAHacc and %PAHdeg were significantly different between non-axenic and axenic cultures (P < 0.05, Table 3). There was more PAH accumulated in the axenic culture (52.8% ± 3.0% for BaP and 33% ± 2.0% for Flt) than in the non-axenic culture (18.1 ± 1.6% for BaP and 11.3 ± 1.5% for Flt). Conversely, the highest degradation was observed in non-axenic cultures for both BaP and Flt (79.4 ± 2.3% and 88.2 ± 4.2%, respectively). Interestingly, the abiotic removal of PAHs, mainly due to the photooxidation, did not account for more than 10 ± 1.8% for BaP and 7.9 ± 1.1% for Flt. Hence, biodegradation was generally the main process of PAH removal. Biodegradation of Flt was higher than that of BaP under both conditions, while more BaP was accumulated. Interestingly, significant differences (P < 0.05) were observed for bacterial and diatom degradation between both PAHs. Bacteria were able to degrade 40.7 ± 2.5% of the initial BaP concentration, whereas the diatom showed a lower ability to degrade this PAH (28.7 ± 2.6%).

In contrast, the microalgae exhibited a high degradation of Flt (53.5 ± 3.2%) compared to the bacteria (26.8 ± 2.2%). Moreover, the degradation level of Flt by the diatom was significantly (P < 0.05) larger than that of BaP.

Diversity and abundance of attached and free-living bacteria communities associated to

Nitzschia sp.

The diversity of attached (AB) and free-living bacteria (FB) associated to Nitzschia sp. was evaluated using the 16S rRNA gene high-throughput sequencing approach. To identify and compare both AB and FB diversity, sequences were analyzed under the same conditions. Overall, 865 different OTUs were detected in the prokaryotic communities associated to the Nitzschia sp.

culture. However, the AB community displayed higher species richness (observed OTUs) ( 562OTUs) and species diversity (Simpson, Shannon indices) than the FB community (303 OTUs) (Table 4). In addition, the average abundance of free-living cells was lower (0.34 ± 0.06 × 10 5 DNA copies ml -1 ) than that of attached bacteria (2.1 ± 0.42 × 10 5 DNA copies mL -1 ).

Analysis of AB community composition showed that Alphaproteobacteria (54.0%), Cyanobacteria (35.5%), Bacteroidetes (3.4%), Gammaproteobacteria (3.3%), and Planctomycetes

(2.7%) dominated the bacterial communities (Fig. 2a). The FB were greatly dominated by Alphaproteobacteria (51.9%), followed by Bacteroidetes (36.6%), Betaproteobacteria (4.8%), Firmicutes (2.0%), and Planctomycetes (1.2%). Within the archaea domain, Euryarchaeota was exclusively presented at low abundance (0.25%) in the AB fraction (Fig. 2a). Alphaproteobacteria (representing more than half of the prokaryote communities) and Planctomyctes (found in lower proportion) were observed in both AB and FB fractions. Actinobacteria, Betaproteobacteria, and Firmicutes were typically found among the FB fraction, while Cyanobacteria and Gammaproteobacteria were exclusively found among the attached bacteria, while Bacteroidetes were more abundant in the FB fraction than in the AB sample.

A Venn diagram demonstrated that OTUs differed among AB and FB fractions (Fig. 2c). In total, 217 OTUs were shared between both samples. A larger number of specific OTUs was detected in the AB fraction (345 OTUs) than in the FB fraction (86 OTUs). However, AB and FB were only dominated by 12 and 14 OTUs, respectively (Table S1). To further analyze the microbial community composition and structure, dominant genera (> 1% of all sequences) are presented in Fig. 2b. The most abundant genera, namely Phaeocystidibacter, Mabikibacter, Roseitalea, Nioella, and Erythrobacter, were identified in both AB and FL fractions. The genera Dulcicalothrix, Chamaesiphon, Roseimaritima, Oricola, and Aestuariibius were greatly related to the AB fraction. The FB fraction was particularly dominated by a member of the family Cryomorphaceae, previously isolated from marine algae (35.5% of all sequences), followed by the genera Agrococcus, Pyruvatibacter, Acuticoccus, Marivita, Roseibaca, and Alcaligenes.

Morphological characterization of bacterial isolates

Based on the morphological characteristics of the colonies, six bacterial strains from diatom Nitzschia sp. pure cultures were isolated and tested for their growth on crude oil (Table 5). Five strains (2, 3, 4, 5, and 6) belonged to the FB fraction, while Strain 1 was obtained from the AB fraction. Colonies were small, large, circular, round, smooth, oval, and approximately between 0.05 and 5 µm of diameter. Among these colonies, three strains (1, 2, 6) forming cream-colored colonies were obtained. Other colonies were yellow (Strain 3), white (Strain 4), and transparent (Strain 5). Cells of aerobic isolates were long rods (3-6 µm long x 0.1 µm wide) or coccoid (0.2-0.5 µm size), occurring as single cells or in pairs.

Phylogenetic analysis of attached and free-living bacterial isolates

The 16S rRNA gene sequences from the six strains were generated to determine their taxonomic group. Phylogenetic analysis revealed that those isolates belonged to the three phyla:

Proteobacteria (Gammaproteobacteria class), Firmicutes, and Actinobacteria (Fig. 3). Strains 1 (attached bacteria), 4, and 6 (free-living bacteria) were related to the genus Staphylococcus. The free-living bacterial Strains 2 and 3 belonged to Acinetobacter and Micrococcus, respectively.

Strain 5 was affiliated to the species Bacillus amyloliquefaciens. These bacterial taxa showed low abundances (< 0.5% of all sequences) in both AB and FB fractions.

Screening for bacteria growing on crude oil

Following isolation and purification, all selected strains were screened for their potential growth in the presence of crude oil. After 15 days of incubation, there was a visual change in the aspect of oil crude with Strains 3, 4, and 6. After 30 days, microscopic observation showed that cultures of these strains had a clear appearance and a turbid aspect, suggesting that Strains 3, 4, and 6 (belonging to the genera Micrococcus and Staphylococcus) could maintain their growth in the presence of crude oil under aerobic conditions (Table 5).

Discussion

Influence of the associated bacteria on Nitzschia growth

In comparison to the axenic condition, Nitzschia sp. exhibited higher growth in non-axenic cultures (Table 1), with higher biomass and cell density (Fig. 1). This result agrees with previous findings that the presence of bacteria can promote the proliferation of microalgae [START_REF] Kazamia | Mutualistic interactions between vitamin B12-dependent algae and heterotrophic bacteria exhibit regulation[END_REF][START_REF] Amin | Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria[END_REF]. A commonly studied interaction between microalgae and their associated bacteria is the bacterial production of vitamins (cobalamin, thiamine, and biotin)) required by algal species for their growth [START_REF] Kazamia | Mutualistic interactions between vitamin B12-dependent algae and heterotrophic bacteria exhibit regulation[END_REF]. Diatom growth can also stimulated by bacteria (mainly Proteobacteria) through the excretion of extracellular polysaccharides (EPS) by phototrophs. These EPS, after bacterial remineralization, constitute an important nutrient source [START_REF] Bruckner | Bacteria associated with benthic diatoms from Lake Constance: Phylogeny and Influences on diatom growth and secretion of extracellular polymeric substances[END_REF]. Other studies also reported that the bacterial presence is beneficial for diatoms through the secretion of extracellular indole acetic acid (IAA), which is an auxin that optimizes algal growth [START_REF] Seyedsayamdost | The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis[END_REF][START_REF] Lépinay | First insight on interactions between bacteria and the marine diatom Hasleaostrearia: Algal growth and metabolomic fingerprinting[END_REF]. [START_REF] Amin | Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria[END_REF] showed that Sulfitobacter species could favor the division of the diatom Pseudonitzschia multiseries via the production of IAA by using the diatom's secreted and endogenous tryptophan.

Tolerance of Nitzschia sp. to PAHs

In this study, Nitzschia sp. was exposed to high levels of PAHs (256 µg Flt L -1 and 3 µg BaP L -1 ), which exceeded the EC50 of growth inhibition reported for diatoms (18-200 µg L -1 for Flt and 1.18 µg L -1 for BaP) [START_REF] Liu | Toxicity of fluoranthene and its biodegradation by Cyclotella caspia alga[END_REF][START_REF] Niehus | Miniaturised marine algae test with polycyclic aromatic hydrocarbons -Comparing equilibrium passive dosing and nominal spiking[END_REF]. Despite this, the diatom kept a continuous proliferation until the end of the incubation in both axenic and non-axenic conditions (Fig. 1). In some case, biomass and cell density in contaminated cultures were lower than in the control, but growth rates were not significantly different, except for BaP in the axenic culture (Table 1; Fig. 1). This indicates that Nitzschia sp. can tolerate both HAPs; this is in agreement with the study of [START_REF] Croxton | The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris[END_REF], who showed that the growth of Nitzschia brevirostris was unaffected by high a concentration of naphthalene (1,000 μg L -1 ). Nevertheless, other studies have observed a significant decrease in diatom growth after PAH exposition [START_REF] Bopp | Gene regulation in the marine diatom Thalassiosira pseudonana upon exposure to polycyclic aromatic hydrocarbons (PAHs)[END_REF][START_REF] Niehus | Miniaturised marine algae test with polycyclic aromatic hydrocarbons -Comparing equilibrium passive dosing and nominal spiking[END_REF]. Obviously, the degree of tolerance/sensitivity to pollutants varies among algal species and depends greatly on the environment in which microalgae evolve (Ben Othmen et al., 2018;[START_REF] Pikula | Toxicity bioassay of waste cooking oil-based biodiesel on marine microalgae[END_REF]. According to [START_REF] Kottuparambil | PAHs sensitivity of picophytoplankton populations in the Red Sea[END_REF], microalgae, via natural evolution, can gain higher resistance to pollutants, allowing the survival of their populations in highly polluted environments. Consequently, Nitzschia sp., isolated from a PAH-contaminated sediment of the Bizerte Lagoon [START_REF] Lafabrie | Contaminated sediment resuspension induces shifts in the structure and the functioning of phytoplankton in an eutrophic Mediterranean lagoon[END_REF][START_REF] Pringault | Consequences of contaminant mixture on the dynamics and functional diversity of bacterioplankton in a southwestern Mediterranean coastal ecosystem[END_REF], seems to be accustomed to high levels of PAHs and has acquired physiological abilities to tolerate and even metabolize these pollutants. This is in line with the idea that several diatom species were identified as good indicators of high PAH levels in surface sediments [START_REF] Potapova | Potential effects of sediment contaminants on diatom assemblages in coastal lagoons of New Jersey and New York States[END_REF]. Interestingly, in PAH treatments, especially for BaP, Nitzschia sp. growth rates were lower in axenic conditions compared to non-axenic ones (Table 1, Fig. 1). This suggests that the presence of bacteria was beneficial for the diatom by improving its capacity to tolerate PAHs, which leads us to infer that associated bacteria could protect the microalgae by reducing PAH toxicity, as has been observed

for metals and pesticides [START_REF] Fouilland | Influence of bacteria on the response of microalgae to contaminant mixtures[END_REF] or tannic acid [START_REF] Bauer | Effects of bacterial communities on the sensitivity of the phytoplankton Stephanodiscus minutulus and Desmodesmus armatus to tannic acid[END_REF].

PAH accumulation and biodegradation

The PAH concentration in the dissolved fraction was extremely low at the end of the incubation, as the amounts of BaP and Flt remaining in the medium did not exceed 2.5-8.5 and 0.5-5.6%, respectively (Table 3). Several processes, including abiotic and biotic factors, can contribute to PAH elimination [START_REF] Ghosal | Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A Review[END_REF]. Abiotic losses, mainly due to photooxidation, did not account for more than 10%. This indicates the high capacity of microorganisms to eliminate PAHs, which is in agreement with previous studies reporting that a large fraction of PAH removal in microalgae culture was attributed to biotic degradation [START_REF] Hong | Accumulation and biodegradation of phenanthrene and fluoranthene by the algae enriched from a mangrove aquatic ecosystem[END_REF][START_REF] Diaz | Evaluation of native acidophilic algae species as potential indicators of polycyclic aromatic hydrocarbon (PAH) soil contamination[END_REF][START_REF] Kumari | Landfill leachate treatment using bacto-algal coculture: An integrated approach using chemical analyses and toxicological assessment[END_REF][START_REF] García De Llasera | Mini-bioreactors with immobilized microalgae for the removal ofbenzo(a)anthracene and benzo(a)pyrene from water[END_REF].

The amounts of PAHs biodegraded by Nitzschia sp. alone (28.7-53.7%) were 1.5-2.4 times lower than when bacteria were present (69.4-80.3%) (Table 3). This suggests co-metabolic synergy between microalgae and bacteria in the removal of PAHs, as previously reported [START_REF] Borde | Synergistic relationships in algal-bacterial microcosms for the treatment of aromatic pollutants[END_REF][START_REF] Coulon | Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats[END_REF][START_REF] Kumari | Landfill leachate treatment using bacto-algal coculture: An integrated approach using chemical analyses and toxicological assessment[END_REF]. The known hydrocarbon-degrading genera Marivita, Erythrobacter, and Alcaligenes (Fig. 2b) and strains growing on crude oil (Table 3) were observed in the bacterial community associated to Nitzschia. This suggests that these associated hydrocarbonoclastic bacteria may enhance biodegradation of PAHs in non-axenic cultures, as previously highlighted [START_REF] Mishamandani | Response of the bacterial community associated with a cosmopolitan marine diatom to crude oil shows a preference for the biodegradation of aromatic hydrocarbons[END_REF][START_REF] Thompson | Enhanced crude oil biodegradation potential of natural phytoplankton-associated hydro-carbonoclastic bacteria[END_REF][START_REF] Thompson | Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH[END_REF][START_REF] Severin | The phytoplankton taxon-dependent oil response and its microbiome: correlation but not causation[END_REF]. The degradation of PAHs by microorganisms depends on their molecular weight, hydrophobicity, and water solubility [START_REF] Haritash | Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review[END_REF]. In general, PAHs with lower molecular weights, lower octanol-water partitioning coefficients (Kow), and higher water solubility are more susceptible to biodegradation. The Flt (four-ring compound) displays a lower molecular weight, a lower log Kow, and a higher water solubility compared to the five-ring BaP (154 vs.

252 g mol -1 , 5.2 vs. 6, and 240 vs. 1.5 µg L -1 , respectively). Biodegradation of Flt by Nitzschia sp.

(53.5%) was higher than that of BaP (28.7%), whereas a higher proportion of BaP remained accumulated in the diatom, as estimated by the PAH concentration measured in the particular phase (Table 3). This suggests that Flt was more easily metabolized by Nitzschia sp. than BaP, which appeared to be more stable and more difficult to degrade. However, in the non-axenic culture, bacterial degradation of BaP (40.7%) was greater than that of Flt (26.8%). Indeed, a bacterial consortium has previously been shown to prefer degrading five-to-six-ring PAHs [START_REF] Moscoso | Efficient PAHs biodegradation by a bacterial consortium at flask and bioreactor scale[END_REF][START_REF] Pugazhendi | Biodegradation of low and high molecular weight hydrocarbons in petroleum refinery wastewater by a thermophilic bacterial consortium[END_REF].

The accumulation of PAHs by the diatom also contributed to their removal, with %PAHacc varying from 11.3 to 52.8 (Table 3). This is in agreement with previous reports for PAH accumulation by microorganisms, including diatoms (such as Skeletonema costatum, Nitzschia sp., Pseudonitzschia spp.) [START_REF] Lei | Removal of fluoranthene and pyrene by different microalgal species[END_REF][START_REF] Hong | Accumulation and biodegradation of phenanthrene and fluoranthene by the algae enriched from a mangrove aquatic ecosystem[END_REF]. The %PAHacc values were higher in axenic (52.8% for BaP and 33% for Flt) than in non-axenic cultures (18.1% for BaP and 11.3%

for Flt) (Table 3). The bioaccumulation of PAHs by microalgae is directly related to the size and the morphology of cells as well as the initial cell inoculum [START_REF] Chan | Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum[END_REF]. In our experiment, the same isolate of Nitzschia sp. was used in both axenic and non-axenic cultures with similar inoculum densities. However, in the non-axenic culture, bacteria were able to degrade BaP and Flt during the incubation, thereby decreasing the amount of PAH that might be accumulated in the diatom cells (Table 3). Besides their degradation, associated bacteria might also influence the availability of BaP and Flt for the diatom through the production of refractory/hydrophobic dissolved organic matter (DOM). Heterotrophic bacteria are known to produce refractory DOM via the assimilation of freshly produced/labile phytoplankton DOM [START_REF] Jiao | Microbial production ofrecalcitrant dissolved organic matter: longterm carbon storage in the global ocean[END_REF][START_REF] Moran | Deciphering ocean carbon in a changing world[END_REF]. This bacteria-derived DOM, more aromatic/hydrophobic, could in turn bind with Flat and BaP through hydrophobic interactions. These DOM-PAHs complexes are larger and more polar than the free dissolved PAHs, thereby decreasing their availability for the diatom [START_REF] Mei | Binding characteristics of perylene, phenanthrene and anthracene to different DOM fractions from lake water[END_REF][START_REF] Yang | Insights into the binding interactions of autochthonous dissolved organic matter released from Microcystis aeruginosa with pyrene using spectroscopy[END_REF]. The capacity of bacteria associated to Nitzschia sp. in reducing PAH accumulation in the diatom and their potential effect on microalgal metabolism may enable the diatom to cope with PAHs, resulting in better growth in non-axenic cultures compared to axenic ones (Table 3, Fig. 1).

Composition of associated bacterial communities and hydrocarbonoclastic potential of bacterial strains

There was a difference in community structure between attached (AB) and free-living (FB) bacteria associated to Nitzschia sp; the AB community exhibited a higher diversity and abundance than the FB community. Additionally, a significant variation in the relative abundance between attached and free-living communities was observed (Table 4, Fig. 2). Many studies indicated prominent differences in the community composition of particle-attached and free-living bacteria [START_REF] Grossart | Marine diatom species harbour distinct bacterial communities[END_REF][START_REF] Bagatini | Host-specificity and dynamics in bacterial communities associated with bloom-forming freshwater phytoplankton[END_REF][START_REF] Rieck | Particle-associated differ from freeliving bacteria in surface waters of the Baltic Sea[END_REF]. The structure of the bacterial community in the phycosphere, both attached and free-living, is specific to algal species, since algae can release strain-dependent organic compounds that are used by specific bacteria [START_REF] Bagatini | Host-specificity and dynamics in bacterial communities associated with bloom-forming freshwater phytoplankton[END_REF]. In this sense, the difference between Nitzschia-AB and FB could be explained by the consistent associations between specific bacterial taxa and the Nitzschia sp. host [START_REF] Amin | Interactions between diatoms and bacteria[END_REF][START_REF] Sison-Mangus | Host-specific adaptation governs the interaction of the marine diatom, Pseudo-nitzschia and their microbiota[END_REF][START_REF] Behringer | Bacterial communities of diatoms display strong conservation across strains and time[END_REF]. Our results revealed that Nitzschiaattached bacteria mainly belonged to Proteobacteria (i.e., Alphaproteobacteria) and Cyanobacteria (Fig. 2a). Previous studies have indicated that Proteobacteria are commonly associated to benthic diatom species [START_REF] Jauffrais | Effect of bacteria on growth and biochemical composition of two benthic diatoms Halamphora coeaeformis and Entomoneis paludosa[END_REF]Keodoodor et al., 2019), and autotrophic nitrogen-fixing bacteria (Cyanobacteria) are known to interact with diatoms [START_REF] Amin | Interactions between diatoms and bacteria[END_REF].

Alphaproteobacteria also predominated in the FB community of Nitzschia sp., as reported previously [START_REF] Grossart | Marine diatom species harbour distinct bacterial communities[END_REF][START_REF] Buchan | Master recyclers: features and functions of bacteria associated with phytoplankton blooms[END_REF]. Bacteroidetes, known as a common bacterial phylum associated to diatoms [START_REF] Grossart | Marine diatom species harbour distinct bacterial communities[END_REF][START_REF] Amin | Interactions between diatoms and bacteria[END_REF][START_REF] Jauffrais | Effect of bacteria on growth and biochemical composition of two benthic diatoms Halamphora coeaeformis and Entomoneis paludosa[END_REF]Keodoodor et al., 2019), formed a large fraction of the FB of Nitzschia sp. (Fig. 2a). Attached and free-living communities were dominated by 16 genera (Fig. 2b), many of which are commonly being observed in algal cultures or as epibionts in several phycospheres of dinoflagellates, diatoms, and coccolithophores [START_REF] Foster | Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses[END_REF][START_REF] Wang | Pyruvatibacter mobilis gen. nov., sp. nov., a marine bacterium from the culture broth of Picochlorum sp. 122[END_REF].

The genera Marivita, Erythrobacter, and Alcaligenes, mainly found in the FB community (Fig. 2b), degrade a wide range of xenobiotics, including aliphatic and polyaromatic hydrocarbons [START_REF] Yuan | The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge[END_REF][START_REF] Durán | Complete genome sequence of the marine hydrocarbon degrader Alcaligenes aquatilis QD168, isolated from crude oil-polluted sediment of Quintero Bay, central Chile[END_REF][START_REF] Severin | The phytoplankton taxon-dependent oil response and its microbiome: correlation but not causation[END_REF]. Furthermore, Strains 3, 4, and 6, isolated from the associated bacterial community, were able to grow in the presence of crude oil (Table 5), suggesting the development of oil degraders in the bacterial community associated to the diatom. Our result corroborated with previous reports on the presence of oil-degrading bacteria in some microalgae phycospheres (Mishamandani et al., 2015;[START_REF] Severin | Dynamics of Heterocapsa sp. and the associated attached and free-living bacteria under the influence of dispersed and undispersed crude oil[END_REF][START_REF] Thompson | Enhanced crude oil biodegradation potential of natural phytoplankton-associated hydro-carbonoclastic bacteria[END_REF][START_REF] Thompson | Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH[END_REF]. Strains 3, 4, and 6 were related to the genera Staphylococcus and Micrococcus, which have previously been identified as PAH degraders [START_REF] Dong | Distribution of PAHs and the PAH-degrading bacteria in the deep-sea sediments of the high-latitude Arctic Ocean[END_REF][START_REF] Ghosal | Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A Review[END_REF][START_REF] Alegbeleye | Bioremediation of polycyclic aromatic hydrocarbon (PAH) compounds: (acenaphthene and fluorene) in water using indigenous bacterial species isolated from the Diep and Plankenburg rivers[END_REF]. Furthermore, species of Micrococcus and Staphylococcus, isolated from contaminated marine sediments, have been identified as naphthalene-degraders [START_REF] Melcher | Impact of irradiation and polycyclic aromatic hydrocarbon spiking on microbial populations in marine sediment for future aging and biodegradability studies[END_REF][START_REF] Zhuang | Importance of Grampositive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments[END_REF] and as biosurfactant producers [START_REF] Ibrahim | Production and partial characterization of biosurfactant produced by crude oil degrading bacteria[END_REF][START_REF] Bao | Lipopeptide biosurfactant production bacteria Acinetobacter sp. D3-2 and its biodegradation of crude oil[END_REF][START_REF] San | Journal of petroleum science and engineering production and structural characterization of biosurfactant produced by newly isolated staphylococcus xylosus STF1 from petroleum contaminated soil[END_REF]. Although associated bacterial communities are generally species-dependent, they can also be influenced by environmental conditions. The benthic Nitzschia sp., used in our study, thrives in PAH-contaminated sediments [START_REF] Lafabrie | Contaminated sediment resuspension induces shifts in the structure and the functioning of phytoplankton in an eutrophic Mediterranean lagoon[END_REF][START_REF] Pringault | Consequences of contaminant mixture on the dynamics and functional diversity of bacterioplankton in a southwestern Mediterranean coastal ecosystem[END_REF], conditions that favor the presence of hydrocarbonoclastic bacteria [START_REF] Ben Said | Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia[END_REF] that might be in close interactions with the benthic diatom. The phycosphere of Nitzchia sp. could have contained hydrocarbonoclastic bacteria (mainly in the FB fraction), which could play an important role in the strong biodegradation of Flt and BaP under non-axenic conditions (Table 3).

Conclusions

The biodegradation, the most significant process of PAH removal, was significantly higher when bacteria were present in the culture of the benthis diatom (Nitzschia sp.). This highlights a possible co-metabolic synergy between microalgae and bacteria in PAH biodegradation, allowing potential bioremediation applications for the removal of PAHs from marine environments.

Investigation of the structure of bacterial communities associated to Nitzschia sp. (the phycosphere) revealed the possible presence of hydrocarbon-degrading bacteria belonging to the genera Marivita, Erythrobacter, and Alcaligenes. Furthermore, the associated bacteria harbored some strains, assigned to Staphylococcus and Micrococcus, which were able to grow on crude oil.

This indigenous diatom, with its associated hydrocarbonoclastic bacteria, could find an interesting application in environmental biotechnology, enhancing the bioremediation of PAH-contaminated sites, or in depollution strategies following oil spills.

Table 1

Exponential growth rate (d -1 ) of Nitzschia sp. grown under non-axenic and axenic conditions in

Figure captions

  CPAHdis / CPAHinitial x 100 %PAHacc= CPAHpart / CPAHinitial x 100 %PAHdeg = (CPAHinitial -CPAHdis-CPAHpart) x 100, where CPAHinitial represents the amount of PAHs added at the beginning of the incubation and CPAHdis and CPAHpart represent the amounts of PAHs measured at the end in the dissolved and particular fractions, respectively.Many processes could contribute to the degradation of PAHs during the incubation. The main abiotic process is photodegradation (PAHpho), but volatilization (PAHvol) and adsorption on the walls of the glass bottles (PAHads) may also lead to PAH decrease and were therefore considered in the calculation. The biotic processes were assigned to the biodegradation by microorganisms (PAHbiodeg), including diatoms (PAHdiadeg) and total bacteria (PAHbacdeg). The importance of these processes is expected to differ among the experimental conditions, and hence, the percentage of degraded PAHs is likely to vary:Sterile conditionin the dark: %PAHdeg = %PAHvol + %PAHads (Eq. 1)

  control (C) and contaminated treatments by two PAHs (BaP and Flt). Values are means ± standard deviations, letters indicate homogenous group, r 2 is coefficient of determination

Fig. 1 .

 1 Fig. 1. Temporal evolution of Chl a concentration and growth kinetics of Nitchizia sp. under nonaxenic (a and b) and axenic (c and d) conditions in control (C) and contaminated treatments by BaP and Flt. Values are means ± standard deviations.

Fig. 2 .

 2 Fig. 2. Relative abundance of microbial communities in attached (AB) and free-living bacteria (FB) from Nitzschia sp. Culture. A. Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with archaea and bacterial phyla or proteobacterial classes in AB and FB fractions. B. Major genera from dominant OTU (>1% of all sequences) in AB and FB fractions. C. Venn diagram showing the distribution of prokaryotic measurable OTUs in AB and FB samples.

Fig. 3 .

 3 Fig. 3. Phylogenetic tree based on similarities of 16S rRNA sequences of bacterial isolates and its relatives. The tree was based on the Juke-Cantor model and the Neighbor-Joining method with bootstrap values for 1000 replicates. The scale bar represents 5% estimated sequence divergence. The archaeal sequence of Natrinema altunense was used as the outgroup

Table 2

 2 Theoretical and measured concentrations of Flt and BaP in Nitzschia sp. cultures under different conditions (axenic, non-axenic and sterile)

	Values are means ± standard deviations
		Theoretical concentration
		Flt (256 µg L -1 )	BaP (3 µg L -1 )
	Measured concentrations
	Axenic	238.11 ± 4.68	2.53 ± 0.42
	Non-axenic	250.79 ± 2.65	2.36 ± 0.27
	Sterile, light	255.68 ± 3.09	2.59 ± 0.43
	Sterile, dark	242.39 ± 4.10	2.65 ± 0.41

Table 3

 3 The % of PAHs (BaP and Flt) accumulated in cells (%PAHacc), remaining in the medium (%PAHdis) and degarded by all processes (%PAHdeg), including abiotic process [photo-oxidation (%PAHpho), volatilization (%PAHvol) and adsorption (%PAHads)] and biodegradation by microorganisms (%PAHbiodeg)[by diatom (%PAHdiadeg) or by bacteria (%PAHbacdeg)], in the Nitzschia sp. culture under non axenic and axenic conditions. Values are mean ± standard deviation.

		BaP		Flt
		non-axenic	axenic	non-axenic	axenic
	%PAHacc	18.1 ± 1.6	52.8± 3.0	11.3 ± 1.5	33.0 ± 2.0
	%PAHdis	2.5 ± 0.6	8.5± 1.4	0.5 ± 0.0	5.6 ± 1.3
	%PAHdeg	79.4 ± 2.3	38.7± 2.0	88.2 ± 4.2	61.4 ± 4.1
	%PAHpho	10.0 ± 1.8	10.0± 1.8	7.9 ± 1.1	7.9 ± 1.1
	%HAPvol+	0	0	0	0
	%PAHads
	%PAHbiodeg	69.4 ± 2.6	28.7 ± 2.6	80.3 ± 5.2	53.5 ± 3.2
	%PAHdiadeg	28.7 ± 2.6	28.7 ±	53.5 ± 3.2	53.5 ±
		2.6		3.2
	%PAHbacdeg	40.7 ± 2.5		26.8 ± 2.2

Acknowledgements

This work was supported by IRD through the Laboratoire Mixte International (LMI) COSYS-Med (Contaminants et Ecosystèmes Sud Méditerranéens). English grammar and syntax of the manuscript were revised by Proof-Reading-Service.com.

1030