Joyce Álvarez Barragán

EXPLORING FUNGI-BACTERIA INTERACTION IN PAH CONTAMINATED COASTAL SEDIMENT

Keywords: fungi, bacteria, microbial diversity, interactions, translocation network, hyphosphere, PAHs, contamination, bioavailability, sediments, aerobic/anaerobic

I.1. Marine sediments

. Marine sediments cover the ̴ 70% of the Earth's surface and comprise the largest single ecosystem on Earth in area. The size of this ecosystem allows a variety of environments from tidally exposed areas, where temperature, salinity, and sediment water content fluctuate greatly, to the greatest depths of the oceans, where no light penetrates, temperatures approach freezing, environmental variables are largely invariant, and pressure exceeds 100 times that experienced at sea level (Snelgrove, 2001). Furthermore, sediments composition is very heterogeneous according to their source of origin (Figure I.1) which are divided in two: lithogenic and biogenic. The lithogenic

General introduction

Sediments occupy around 70% of the area of the planet, making them the biggest ecosystem on Earth.

Beside their extension, several factors vary between sediment locations that can go from the coastal sediments to the bottom of the deep sea; this confers sediments a variety of physicochemical characteristics that include salinity, oxygen concentration, particle size, content of organic matter and many more. With all of these heterogenic characteristics, it is not surprising to find a vast microbial diversity inhabiting these ecosystems. The microorganisms that live in sediments and their interactions play a key role in several geochemical processes including sulphur, nitrogen and carbon cycling. Nevertheless, the dynamics of microorganisms and their interactions are constantly affected by the input of hydrocarbon contamination due to natural or anthropogenic sources.

From the hydrocarbons that enter in the marine environment, polycyclic aromatic hydrocarbons (PAHs) are pollutants of main concern due to their toxic, mutagen and carcinogenic potential.

Moreover, the transformation products of incomplete degradation of PAHs (generally by physical or chemical processes) are more toxic than parent PAHs, and lead to critical cellular effects. For this reason, the microbial biodegradation that involves the transformation of PAHs in carbon dioxide and water has become an alternative to cleaning PAHs polluted sediments.

Several microorganisms have been identified and characterized for their capacity to tolerate and degrade PAHs. From these microorganisms, fungi and bacteria have been studied, and the pathways they use to degrade PAHs have been described. Fungi degrade PAHs extracellularly using ligninolytic monooxygenase enzymes or intracellularly via P450 cytochrome. Whereas, bacteria have PAHs degradation pathways that change depending on the presence of oxygen (aerobic pathway) or the absence of it (anaerobic pathway). Nevertheless, fungi and bacteria have their limitations. To date, there is no report of fungi that can mineralize completely PAHs to carbon and water, and bacteria cannot degrade high molecular weight PAHs efficiently. This is why there is interest to focus on the interaction of fungi and bacteria together for the complete biodegradation of PAHs. In hydrocarbonpolluted sediments, fungi are likely primary degraders of high molecular weight hydrocarbons via secreted extracellular enzymes, and work synergistically with hydrocarbon-degrading bacteria. The interaction of fungi and bacteria give one another special advantages to overcome PAHs pollution. It is described that bacteria can improve fungal reproduction and affect their metabolism. Besides, fungi facilitate bacterial growth and shape bacterial communities. Moreover, fungi and bacteria collaborate for PAHs degradation. Fungal mycelia mobilize intracellularly entrapped PAHs making them bioavailable for bacterial degradation and bacteria use fungal hyphae as paths for dispersion. The dispersion of bacteria by fungal mycelia is highly relevant in sediments where PAHs accumulate creating hydrophobic patches that are more resistant to degradation. However, no information exists on the effect of fungal transport of bacteria in PAHs contaminated marine sediments.

In this context, the work presented here shows the effort made to understand fungal communities and their interactions with bacterial communities from PAHs contaminated coastal sediments; making an emphasis on the impact of PAHs on fungal and bacterial diversity. To accomplish this, a bibliographic synthesis of the current knowledge is presented, including the role of fungi in marine environment, their potential to biodegrade PAHs and their interactions with bacterial communities for PAHs degradation (Chapter I). The results of the thesis research are presented in chapters in scientific publication format, already submitted or published, starting with the impact of PAHs contamination on fungal and bacterial communities diversity from coastal sediments and their inter-kingdom interactions. These interactions were evaluated constructing co-occurrences networks between fungi and bacteria from coastal sediment with no contaminated samples and comparing them with networks constructed with contaminated samples (Chapter II). Following, it was done the isolation of fungal cultivable diversity of coastal sediments, which were evaluated of PAHs removal capacity, and the presence of the genes related to PAHs degradation (Chapter III). Finally, two fungi selected for their lowest and highest removal capacities were used to analyze fungal selectivity over bacterial communities and their bacterial translocation potential through contaminated sediment gaps in order to find biomarkers of fungal and bacterial interactions in PAHs contaminated sediments (Chapter IV).

The analysis of results and further perspectives of thesis research are presented in the last chapter (Chapter V). The comprehension and elucidation of fungal and bacterial interactions in PAHs contaminated sediments presented might give important knowledge for further implementation of bioremediation strategies to mitigate the effect of PAHs in contaminated environments.

Introduction générale

Les sédiments représentent environ 70 % de la superficie de la planète, ce qui en fait le plus grand écosystème sur Terre. Plusieurs facteurs varient en fonction de leur emplacement, allant de la côte jusqu'aux abysses, conférant aux sédiments diverses caractéristiques physico-chimiques, comme la salinité, la concentration en oxygène, la taille des particules, la teneur en matière organique et bien d'autres. Il n'est alors pas surprenant de trouver une vaste diversité microbienne habitant ces écosystèmes. Les micro-organismes qui vivent dans les sédiments et leurs interactions jouent un rôle clé dans plusieurs processus biogéochimiques, notamment dans les cycles du soufre, de l'azote et du carbone. Néanmoins, la dynamique des micro-organismes et leurs interactions sont constamment affectées par l'apport de contaminations aux hydrocarbures, provenant de sources naturelles ou anthropiques.

Parmi les hydrocarbures entrant dans le milieu marin, les hydrocarbures aromatiques polycycliques (HAP) sont des polluants préoccupants en raison de leur potentiel toxique, mutagène et cancérigène.

De plus, les produits de transformation de la dégradation incomplète des HAP (généralement par des processus physiques ou chimiques) sont plus toxiques conduisant à des effets cellulaires critiques.

Pour cette raison, la biodégradation microbienne qui implique la transformation des HAP en dioxyde de carbone et en eau, est devenue une alternative à la remédiation des sédiments pollués par les HAP.

Plusieurs microorganismes ont été identifiés et caractérisés pour leur capacité à tolérer et à dégrader les HAP. Parmi ces microorganismes, des champignons et des bactéries ont été étudiés et leurs voies de dégradation ont été décrites. Les champignons dégradent les HAP de manière extracellulaire à l'aide d'enzymes monooxygénases ligninolytiques ou de manière intracellulaire via le cytochrome P450.

Alors que les bactéries ont des voies de dégradation des HAP qui changent en fonction de la présence (voie aérobie) ou absence (voie anaérobie) d'oxygène. Néanmoins, les champignons et les bactéries ont leurs limites. À ce jour, il n'y a aucune étude rapportant que les champignons sont capables de minéraliser complètement les HAP en dioxyde de carbone et en eau, alors que les bactéries ne peuvent pas dégrader efficacement les HAP de haut poids moléculaire. Ainsi, l'interaction entre champignons et bactéries mérite d'être examinée pour obtenir la biodégradation complète des HAP. Dans les sédiments pollués par des hydrocarbures, les champignons sont probablement des dégradeurs primaires de molécules de poids moléculaire élevé via des enzymes extracellulaires, travaillant en synergie avec les bactéries dégradant les hydrocarbures. L'interaction entre champignons et bactéries donne des avantages particuliers pour surmonter la présence de HAP. Il est décrit que les bactéries peuvent améliorer la reproduction des champignons et affecter leur métabolisme. Par ailleurs, les champignons facilitent la croissance bactérienne et façonnent les communautés bactériennes. De plus, les champignons et les bactéries collaborent pour la dégradation des HAP. Les mycéliums fongiques mobilisent les HAP piégés à l'intérieur de la cellule, les rendant biodisponibles pour la dégradation bactérienne, alors que les bactéries utilisent les hyphes fongiques comme voies de dispersion. La dispersion des bactéries par les mycéliums fongiques est très pertinente dans les sédiments où les HAP s'accumulent créant des zones hydrophobes plus résistantes à la dégradation. Cependant, aucune information n'est disponible sur l'effet du transport fongique des bactéries dans les sédiments marins contaminés par les HAP.

Dans ce contexte, les travaux présentés ici montrent l'effort fourni pour comprendre les communautés fongiques et leurs interactions avec les communautés bactériennes issues des sédiments côtiers contaminés par les HAP ; mettant l'accent sur l'impact des HAP sur la diversité fongique et bactérienne.

Pour ce faire, une synthèse bibliographique des connaissances actuelles est présentée, incluant le rôle des champignons en milieu marin, leur potentiel de biodégradation des HAP et leurs interactions avec les communautés bactériennes pour la dégradation des HAP (Chapitre I). Les travaux de thèse sont présentés dans des chapitres au format de publication scientifique, déjà soumis ou publiés, en commençant par l'impact de la contamination par les HAP sur la diversité des communautés fongiques et bactériennes des sédiments côtiers et leurs interactions. Ces interactions ont été évaluées en comparant des réseaux de co-occurrences entre champignons et bactéries de sédiments côtiers contaminés par des HAP à ceux obtenus de sédiments non contaminés (Chapitre II). Ensuite, il a été fait l'isolement de la diversité fongique cultivable des sédiments côtiers qui ont été évalués de la capacité d'élimination des HAP et de la présence des gènes liés à la dégradation des HAP (Chapitre III).

Enfin, deux champignons sélectionnés pour leurs capacités d'élimination des HAP, l'un ayant la plus faible et l'autre la plus élevée, ont été utilisés pour étudier les interactions champignon-bactéries. Le potentiel de translocation bactérienne des champignons à travers des obstacles de sédiments contaminés a été examiné afin de i) montrer la sélectivité fongique sur les communautés bactériennes et ii) déterminer des biomarqueurs des interactions fongiques et bactériennes dans les sédiments contaminés par les HAP (chapitre IV). Les principales conclusions issues des résultats obtenus et les perspectives de recherche de thèse sont présentées dans le dernier chapitre (Chapitre V). La compréhension et l'élucidation des interactions entre champignons et bactéries dans les sédiments contaminés par les HAP apportent des connaissances importantes pour la mise en oeuvre de stratégies de bio-remédiation visant à atténuer l'effet des HAP dans les environnements contaminés. [START_REF] Maslin | ISOTOPES IN MARINE SEDIMENTS[END_REF].

component is composed of fragments of rocks from different sizes that can go from microscopic clays to large boulders. The biogenic component originates in surface waters and dwells down to deposit; it is composed at least 30% of skeletal debris from microorganisms rich in calcium carbonate, biogenic silica and organic matter [START_REF] Maslin | ISOTOPES IN MARINE SEDIMENTS[END_REF]. With all these heterogeneity of physicochemical parameter, it is not surprising that marine sediments host a wide diversity of microorganisms.

It is estimated that subsea floor constitute almost five-sixths of the total biomass of the planet and onethird of its living biomass [START_REF] Hoshino | Global diversity of microbial communities in marine sediment[END_REF][START_REF] Kallmeyer | Global distribution of microbial abundance and biomass in subseafloor sediment[END_REF][START_REF] Whitman | Prokaryotes: The unseen majority[END_REF]. They play a key role in geochemical processes cycling sulphur, nitrogen, iron and organic or inorganic carbon [START_REF] Parkes | A review of prokaryotic populations and processes in sub-seafloor sediments, including biosphere:geosphere interactions[END_REF]. Being sediments the reservoir of the largest pool of organic carbon on the planet [START_REF] Parkes | A review of prokaryotic populations and processes in sub-seafloor sediments, including biosphere:geosphere interactions[END_REF], it is highly relevant to investigate and analyse the microbial communities inhabiting this ecosystem and their interactions. From these interactions, fungi and their associated bacteria have a key role in the carbon cycling and minerals [START_REF] Verrecchia | Fungi and Sediments[END_REF], highlighting their importance for these ecosystems.

I.2. Diversity of fungi and role in marine sediments

I.2.1. Fungal diversity in marine ecosystems

Fungi occur in different marine environments that include the water bodies in the ocean known as marine-derived fungi [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 marine-derived fungus Cladosporium sp. CBMAI 1237[END_REF][START_REF] Campeão | The Deep-Sea Microbial Community from the Amazonian Basin Associated with Oil Degradation[END_REF][START_REF] Vieira | Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process[END_REF], to coastal sediments [START_REF] Elshafie | Biodegradation of crude oil and n-alkanes by fungi isolated from Oman[END_REF][START_REF] Simister | Degradation of oil by fungi isolated from Gulf of Mexico beaches[END_REF][START_REF] Babu | Fungi Diversity in Different Coastal Marine Ecosystem along South East Coast of India[END_REF][START_REF] Amer | Characterization of some fungal strains isolated from the Eastern coast of Alexandria, Egypt, and some applications of Penicillium crustosum. Egypt[END_REF], mangrove (Ghizelini et al., 2018), deep sea sediments (Redóu et al., 2014;Xu et al., 2014;[START_REF] Edgcomb | Marine subsurface eukaryotes: the fungal majority: Marine subsurface eukaryotes[END_REF][START_REF] Zhang | Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation[END_REF][START_REF] Campeão | The Deep-Sea Microbial Community from the Amazonian Basin Associated with Oil Degradation[END_REF] and even in high artic sediments [START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF].

Fungi can be obligate marine fungi if they grow and reproduce exclusively in marine or estuarine environment (Kohlmeyer and Kohlmeyer 1979), to consider a fungus marine, its growth and reproduction in marine habitat has to be demonstrated (Shaerer et al., 2007). Nevertheless, there are fungi exclusive from these habitats; several families are shared with soil and other environments. Some fungal population in sediment has been described and analysed (Table I.1), but part of their large diversity remains unknown. As first approach, cultivation was the main tool to characterize and identify fungi in marine ecosystems. These studies showed prevalence in strains of the phylum Ascomycota [START_REF] Elshafie | Biodegradation of crude oil and n-alkanes by fungi isolated from Oman[END_REF][START_REF] Simister | Degradation of oil by fungi isolated from Gulf of Mexico beaches[END_REF]Redóu et al., 2014;Ahumada-Rudolph et al., 2016;[START_REF] Mouton | Diversity and Characterization of Culturable Fungi from Marine Sediment Collected from St. Helena Bay, South Africa[END_REF]Ravalet et al., 2000;[START_REF] Babu | Fungi Diversity in Different Coastal Marine Ecosystem along South East Coast of India[END_REF][START_REF] Amer | Characterization of some fungal strains isolated from the Eastern coast of Alexandria, Egypt, and some applications of Penicillium crustosum. Egypt[END_REF][START_REF] Chikere | Characterization of hydrocarbon utilizing fungi from hydrocarbon polluted sediments and water[END_REF]Birolli et al., 2017;Ghizelini et al., 2018). In addition, the phylum Basidiomycota was also found and isolated (Redóu et al., 2014;Ahumada-Rudolph et al., 2016;[START_REF] Mouton | Diversity and Characterization of Culturable Fungi from Marine Sediment Collected from St. Helena Bay, South Africa[END_REF][START_REF] Vieira | Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process[END_REF] and even some strains of Zygomycota (Ravalet et al., 2000;[START_REF] Babu | Fungi Diversity in Different Coastal Marine Ecosystem along South East Coast of India[END_REF][START_REF] Chikere | Characterization of hydrocarbon utilizing fungi from hydrocarbon polluted sediments and water[END_REF]. Nevertheless, with new technologies emerging for sequencing and identification of microorganisms, the exploration of deeper fungal diversity study has been possible. As in culture studies, Ascomycota result in the prevalent phylum (Xu et al., 2014;[START_REF] Zhang | Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation[END_REF][START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF]. In less proportion, Zygomycota [START_REF] Zhang | Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation[END_REF][START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF] and Basidiomycota phyla (Xu et al., 2014;[START_REF] Edgcomb | Marine subsurface eukaryotes: the fungal majority: Marine subsurface eukaryotes[END_REF][START_REF] Zhang | Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation[END_REF][START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF] have been found. More recently, fungi from the phylum Chrytidiomycota have been found and described as one of the most abundant fungal phylum in marine environments [START_REF] Gleason | Zoosporic true fungi in marine ecosystems: a review[END_REF][START_REF] Longcore | Chytridiomycota. eLS[END_REF] (Table I.1). The fungi belonging to Chrytridiomycota are zoosporic fungi; they have singular characteristics including motile spores and association with plankton [START_REF] Grossart | Aquatic fungi: targeting the forgotten in microbial ecology[END_REF]. The finding and classification of new phyla of fungi suggest that more research has to take place to map and evaluate the diversity of this Kingdom and their role in sediment ecosystems.

I.2.2. Role and importance of fungi in marine sediments

The active ecological role of fungi has been overlooked considering that sediment microbial communities are connected by synergistic, antagonistic or neutral relationships. Fungal microbiome plays a role in the overall coordination and whole microbial communities, controlling the structure of sediment microbial functional networks (Booth et al., 2019).

In the marine habitat, fungi interact and associate with almost all marine organisms [START_REF] Ainsworth | The Other Microeukaryotes of the Coral Reef Microbiome[END_REF][START_REF] Yarden | Fungal association with sessile marine invertebrates[END_REF]. In the water column, fungi from Chytridiomycota phylum parasite phytoplankton, diatoms and cyanobacteria contributing to the cycling of organic compounds known as "mycoloop" [START_REF] Frenken | Integrating chytrid fungal parasites into plankton ecology: research gaps and needs: Research needs in plankton chytridiomycosis[END_REF][START_REF] Haraldsson | Microbial parasites make cyanobacteria blooms less of a trophic dead end than commonly assumed[END_REF][START_REF] Kagami | Parasitic chytrids: their effects on phytoplankton communities and food-web dynamics[END_REF]

(Figure I.2).
They modify the amount and composition of the dissolved organic carbon liberated by phytoplankton impacting the structures of bacterial communities [START_REF] Senga | Influence of parasitic chytrids on the quantity and quality of algal dissolved organic matter (AOM)[END_REF] and sediment composition. This is highly relevant because photosynthetic phytoplankton convert dissolved organic carbon to organic matter producing oxygen creating marine webs for other fungi and heterotrophic bacteria [START_REF] Worden | Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes[END_REF]. [START_REF] Grossart | Aquatic fungi: targeting the forgotten in microbial ecology[END_REF].

Fungi have been also found to dominate ocean sediments, particularly in rich-organic matter sediments (Orsi et al., 2013a). In sediments, fungi have been found actively engaged in processing a range of different organic matter types including lipids, proteins, and carbohydrates via specific hydrolases [START_REF] Orsi | Gene expression in the deep biosphere[END_REF]. Deeper down in subsurface sediments, fungal gene expression was associated with growth, division, and sporulation, catalytic activities, and the synthesis of antimicrobial products [START_REF] Pachiadaki | Fungal and Prokaryotic Activities in the Marine Subsurface Biosphere at Peru Margin and Canterbury Basin Inferred from RNA-Based Analyses and Microscopy[END_REF]. These data suggest nutrient recycling and crossfeeding interactions between fungi and other components of the microbial communities [START_REF] Gutiérrez | The role of fungi in processing marine organic matter in the upwelling ecosystem off Chile[END_REF].

In the deep-sea sediments, fungi trap soil and humid material particles acting as a binding agent using polysaccharides [START_REF] Aspiras | Aggregate stabilization by filamentous microorganisms[END_REF][START_REF] Tisdall | Fungal hyphae and structural stability of soil[END_REF] to form microaggregates [START_REF] Kandeler | Response of soil microbial biomass, urease and xylanase within particle size fractions to long-term soil management[END_REF][START_REF] Molope | The Contributions of Fungi, Bacteria and Physical Processes in the Development of Aggregate Stability of a Cultivated Soil[END_REF]. This process trap and cycles the dissolved and particulate organic matter [START_REF] Verdugo | The oceanic gel phase: a bridge in the DOM-POM continuum[END_REF] helping microorganisms like bacteria or other fungi to remain protected in certain particle size [START_REF] Suberkropp | Application of fungal and bacterial production methodologies to decomposing leaves in streams[END_REF]. The presence of large formation of aggregates with fungal filamentous networks sequestrate and store carbon [START_REF] Bailey | Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration[END_REF]. Fungi in the deep sea sediments have a close relationship with chemoautotrophic prokaryotes involved in iron oxidation [START_REF] Bengtson | Deep-biosphere consortium of fungi and prokaryotes in Eocene subseafloor basalts[END_REF]. It is suggested that this association, chemoautotrophs fix the dissolved carbon from fluids and build biomass used by fungi for their metabolism. In turn, fungi metabolize carbohydrates producing CO2 making it available for chemoautotrophs [START_REF] Bengtson | Deep-biosphere consortium of fungi and prokaryotes in Eocene subseafloor basalts[END_REF].

The most studied fungi are aerobic but anaerobic fungi are also known to habit in lakes, landfilled sites [START_REF] Mcdonald | The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters: Anaerobic cellulose degradation in landfill[END_REF], and deep-sea [START_REF] Nagano | Fungal diversity in deep-sea extreme environments[END_REF]. Anaerobic fungi have no mitochondria and are unable to produce energy with anaerobic/aerobic respiration [START_REF] O'fallon | Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188[END_REF][START_REF] Yarlett | Hydrogenosomes in the rumen fungus Neocallimastix patriciarum[END_REF]. As a substitute, they contain organelles called hydrogenosomes that are able to metabolise glucose to cellular energy. This metabolic pathway lead as residues H2, CO2, acetylate, formate, lactate and ethanol (Brul and Stumm, 1994;[START_REF] Yarlett | Hydrogenosomes in the rumen fungus Neocallimastix patriciarum[END_REF]Khejornsart and Wanapat 2010) that are profited by other organisms. For example, the sub-seafloor is the habitat of anaerobic communities that use formate and hydrogen as electron sources [START_REF] Orcutt | Microbial Ecology of the Dark Ocean above, at, and below the Seafloor[END_REF].

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Fungi are thriving, abundant, active, and functioning components of the oceans, from surface sunlit water to deep subsurface sediments and crust, influencing marine biogeochemical cycles in multiple ways. Nevertheless, these cycles and their equilibrium are susceptible to disturbance provoked by pollution, being oil splits and hydrocarbon contamination persistent in these environments. Studying alterations that hydrocarbon pollution cause to fungal diversity apport crucial information to evaluate their impact and develop environmental recovery strategies.

I.3. Degradation of PAHs in marine sediments

I.3.1. PAHs definition and occurrence in marine sediments

Polycyclic aromatic hydrocarbons (PAHs) constitute a class of hazardous organic chemicals consisting of two or more fuzzed rings (Figure I.3). In nature, they are formed during forest fires, volcanic eruptions, or by plant and bacterial reactions (Wilson and Jones, 1993). PAHs formation result also from incomplete combustion of organic materials (through pyrolysis and pyrosynthesis) like coal, tar, oil and gas, automobile exhaust, tobacco and smoked food, either during industrial and other human activities or during geothermal reactions associated with the production of fossil-fuels and minerals, [START_REF] Ghosal | Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review[END_REF]. An estimated 1. [START_REF] Ghosal | Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review[END_REF].

Due to their hydrophobic nature, most PAHs in aquatic and terrestrial ecosystems bind to particles in soil and sediments, rendering them less available for biological uptake, and they also bio-accumulate in food chains (Sanglard et al., 1986). The more rings PAHs have in their structure, recalcitrance increases. Depending on their hydrophobicity, PAHs tend to associate with both suspended particulate materials (SPM) at the water column as well as button sediment, which is recognized as the destination where some contaminants will be deposited and remain for long period of time (Cardoso et al. PAHs occur in various ecosystems and are priority pollutants due to their potential toxicity, mutagenicity, and carcinogenicity. Low-molecular-weight PAHs are acutely toxic and most highmolecular-weight PAHs are mutagenic and carcinogenic [START_REF] Santarelli | Processed Meat and Colorectal Cancer: A Review of Epidemiologic and Experimental Evidence[END_REF]. In addition, the transformation products of PAHs are more toxic than parent PAHs and lead to critical cellular effects [START_REF] Schnitz | Time-varying conjugation of 7,12dimethylbenz(a)antracene metabolites in a rainbow trout (Oncorhynchus mykiss)[END_REF]. Consequently, cleaning of PAHs-polluted places has been thought to be one of the most essential alternatives for restoring environmental damage [START_REF] Ghosal | Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review[END_REF].

Biodegradation of crude oil to carbon dioxide and water is the major process by which hydrocarboncontaminated environments are remediated.

Fungi in PAHs degradation

Fungi appear to play an important role in cycling anthropogenic sources of carbon. Fungi were found to dominate benthic communities impacted by oil and included taxa known to degrade hydrocarbons [START_REF] Bik | Dramatic Shifts in Benthic Microbial Eukaryote Communities following the Deepwater Horizon Oil Spill[END_REF] due to their high tolerance to these compounds [START_REF] Nasrawi | Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico[END_REF]. There are several genera linked to hydrocarbon degradation in sediments [START_REF] Walker | Some effects of petroleum on estuarine and marine microorganisms[END_REF]) such as:

Cladosporium, Cunninghamella, Penicillium, Fusarium, Alternaria, and Aspergillus [START_REF] Nasrawi | Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico[END_REF][START_REF] Passarini | Marine-derived filamentous fungi and their potential application for polycyclic aromatic hydrocarbon bioremediation[END_REF][START_REF] Steliga | Changes in toxicity during in situ bioremediation of weathered drill wastes contaminated with petroleum hydrocarbons[END_REF]) (Table I.2). Fungal PAHs degradation mainly involves monooxygenase enzymes that are intracellular or extracellular. Intracellular PAHs degradation has been characterized in non-ligninolytic fungi, in which cytochrome P450 monooxygenase oxidize PAHs to epoxides and dihydrodiols (Cerniglia and Sutherland, 2010) (Figure I.5). The result of this degradation pathway create secondary compounds that act as potent carcinogens, some times more potent than the same PAHs [START_REF] Cerniglia | Degradation of Polycyclic Aromatic Hydrocarbons by Fungi[END_REF]. For example, degradation that include photochemical reactions can turn PAHs into nitro-PAHs which are carcinogenic [START_REF] Finlayson-Pitts | Tropospheric air pollution: Ozone, airborne toxics, polycyclic aromatic hydrocarbons, and particles[END_REF]. In comparison, the extracellular PAHs degradation (characterized in white rot fungi) involves the production of oxidative extracellular enzymes including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase that transform PAHs in simpler molecules that are used by other organisms as carbon source (Figure I.5). This is why oxidation on PAHs by ligninolytic enzymes represent a better strategy for cleaning contaminated PAHs environment. In addition, ligninolytic enzymes present other advantages: i) as they are secreted extracellularly they are able to diffuse toward immobile PAHs, and ii) they are able to transform a wide range of substrates thanks to their broad substrate specificity [START_REF] Cerniglia | Degradation of Polycyclic Aromatic Hydrocarbons by Fungi[END_REF]. Fungi mineralize PAHs by a combination on ligninolytic enzymes, cytochrome P450 monooxygenase, and epoxide hydrolases [START_REF] Bezalel | Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus[END_REF]. However, most fungi are not able to use PAHs as sole carbon source but they are able to co-metabolize them in more oxidized products making them more available for further degradation by other organisms like bacteria [START_REF] Ghosal | Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review[END_REF].

I.3.4. Bacteria in PAHs degradation

The microbial response to an oil spill at sea is dependent on numerous factors, including the oil composition and degree of weathering, as well as environmental conditions, particularly temperature and nutrient concentrations (Duran and Cravo-Laureau 2016). There are some typical patterns; the most notable is the large increase in abundance of Alcanivorax spp., which degrade straight-chain and branched alkanes [START_REF] Coulon | Effects of temperature and biostimulation on oil-degrading microbial communities in temperate estuarine waters[END_REF][START_REF] Harayama | Microbial communities in oil-contaminated seawater[END_REF][START_REF] Head | Marine microorganisms make a meal of oil[END_REF][START_REF] Kostka | Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill[END_REF]McGenity et al., 2012;[START_REF] Mckew | Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria[END_REF][START_REF] Teramoto | Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment[END_REF][START_REF] Yakimov | Obligate oil-degrading marine bacteria[END_REF] and has a multitude adaptations to access oil and to survive in open marine environments [START_REF] Sabirova | Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borkumensis SK2[END_REF][START_REF] Schneiker | Genome sequence of the ubiquitous hydrocarbondegrading marine bacterium Alcanivorax borkumensis[END_REF] , followed by Cycloclasticus spp., which degrade PAHs [START_REF] Cui | Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge[END_REF][START_REF] Harayama | Microbial communities in oil-contaminated seawater[END_REF][START_REF] Head | Marine microorganisms make a meal of oil[END_REF][START_REF] Teira | Dynamics of the hydrocarbon-degrading Cycloclasticus bacteria during mesocosm-simulated oil spills[END_REF][START_REF] Yakimov | Obligate oil-degrading marine bacteria[END_REF] with key enzymes including alkane hydroxylases and three cytochrome P450-dependent alkane monooxygenases [START_REF] Schneiker | Genome sequence of the ubiquitous hydrocarbondegrading marine bacterium Alcanivorax borkumensis[END_REF].

Another bacteria include strains affiliated to Acinetobacter spp., which are commonly isolated from oilcontaminated marine environments [START_REF] Ron | Acinetobacter and Alkanindiges[END_REF]. They also have a diverse array of alkane hydroxylase systems enabling them to metabolize both short-and long-chain alkanes (van [START_REF] Van Beilen | Alkane hydroxylases involved in microbial alkane degradation[END_REF][START_REF] Rojo | Degradation of alkanes by bacteria[END_REF]. Alcanivorax spp. are sometimes outcompeted by Thalassolituus spp. in temperate environments [START_REF] Mckew | Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria[END_REF], which is classified as obligate hydrocarbondegrading bacteria reaching 90% of the microbial community near an oil spill [START_REF] Yakimov | Obligate oil-degrading marine bacteria[END_REF].

Due to the incidence in the environment Alcanivorax spp. is considered as potential producer of antibacterial molecules (McGenity et al., 2012). Additionally, two NaCl-dependent hydrocarbondegrading from marine environment have been isolated: Planomicrobium alkanoclasticum MAE2 [START_REF] Engelhardt | Isolation and characterization of a novel hydrocarbon-degrading, Gram-positive bacterium, isolated from intertidal beach sediment, and description of Planococcus alkanoclasticus sp[END_REF] and Yeosuana aromativorans GW1-1 [START_REF] Kwon | Yeosuana aromativorans gen. nov., sp. nov., a mesophilic marine bacterium belonging to the family Flavobacteriaceae, isolated from estuarine sediment of the South Sea, Korea[END_REF], which degrade alkanes and PAHs respectively. Other genera, including Acinetobacter, Marinobacter, Pseudomonas and Rhodococcus spp. [START_REF] Duran | Marinobacter[END_REF][START_REF] Gerdes | Influence of crude oil on changes of bacterial communities in Arctic sea-ice[END_REF][START_REF] Vila | Microbial community structure of a heavy fuel oil-degrading marine consortium: linking microbial dynamics with polycyclic aromatic hydrocarbon utilization: Community structure of a marine oil-degrading consortium[END_REF][START_REF] Yu | Degradation of polycyclic aromatic hydrocarbons by a bacterial consortium enriched from mangrove sediments[END_REF], contribute to hydrocarbon degradation.

Sediments add to the complexity of identifying the main hydrocarbonoclastic microbes, but nearly all of the above genera are detected in the aerobic zone of marine sediments and presumed to be active in hydrocarbon degradation. Microbial communities from coastal sediments vary more from one location to another than those from open waters, and have much greater community evenness [START_REF] Zinger | Global Patterns of Bacterial Beta-Diversity in Seafloor and Seawater Ecosystems[END_REF]. Moreover, in sediments, cells are much more concentrated, resulting in a greater likelihood of interactions, which becomes even more prevalent in biofilms where cells are more densely packed (McGenity et al., 2012).

Bacterial PAHs degradation involves two different pathways according to the oxygen availability and presence. In the aerobic catabolism, the initial step involves the oxidation of PAHs to dihydrodiol by a multicomponent enzyme system. This hydroxylated intermediate may then be processed to a central intermediate catechol or protocatechuate, which is cleaved through the ortho or meta cleavage type of pathways. In polynuclear aromatic compounds like PAHs, there is sequential cleavage of the rings via Comparatively, under anoxic (anaerobic) conditions where molecular oxygen is not present, requires alternative terminal electron acceptors like nitrate (NO3 -), sulphate (SO4 2-), manganese (IV), CO2 and Ferric iron (Fe 3+ ) etc. [START_REF] Meckenstock | Anaerobic degradation of polycyclic aromatic hydrocarbons[END_REF]. It has been reported that Sulphate-reducing, denitrifying, and methanogenic bacterial communities contribute to the anaerobic degradation pathway of PAHs (Haritash and Kaushik, 2009) (Figure I.5). As in the aerobic pathway, the anaerobic metabolism requires prior PAH molecule activation. The activation via enzymatic pathways has been described involving a direct carboxylation or a methylation followed by the addition of fumarate. In both cases, another activation step occurs via coenzyme A before the β-oxidation metabolism (Duran and Cravo-Laureau, 2016;Meckenstock et al., 2016). [START_REF] Shahsavari | Phytoremediation and Necrophytoremediation of Petrogenic Hydrocarbon-Contaminated Soils[END_REF].

I.4. Challenges of PAHs biodegradation in marine sediments

As described previously, sediments have unique physical-chemical characteristics, which drive to particular challenges related to PAHs degradation. Sediments are thus hazardous environments where microbial processes involved in PAHs depletion take place.

I.4.1. Oxygen concentration

Thermodynamically, O2 represents the most favourable abundant electron acceptor available for PAH degradation. However, seawater contains little O2, rapidly depleted by microbial activity, generally only extends a few millimetres or centimetres into the sediment. Even in near-surface coastal sediments, oxygen is usually rapidly removed due to aerobic respiration and this thus facilitates anaerobic respiration which uses a sequential series of electron acceptors providing decreasing energy yield (NO 3-, Mn 4+ , Fe 3+ , SO4 2 -, and CO2), and the formation of characteristic zones of NH 4+ , Mn 2+ , Fe 2+ , S 2 -(often as metal sulphides) and CH4 with increasing depth [START_REF] Parkes | Biogeochemistry and biodiversity of methane cycling in subsurface marine sediments (Skagerrak, Denmark)[END_REF].

Biodegradation of hydrocarbons in anoxic marine sediments is slower than in oxic zones [START_REF] Shin | Oxygen demand and sulfate reduction in petroleum hydrocarbon contaminated salt marsh soils[END_REF] and aerobic respiration is considered as primary mechanism of hydrocarbon degradation, even in marine sediments (McGenity et al., 2012). The complete mechanism of PAHs depletion in marine ecosystems is not fully understood, but it is known that aerobic and anaerobic microorganisms coexist and create chemostatic interactions (Gerrits and Gottschal, 1993;[START_REF] Wimpenny | Growth of mixed cultures ofParacoccus denitrificans andDesulfovibrio desulfuricans in homogeneous and in heterogeneous culture systems[END_REF]. In these interactions, the result of respiration of one organism act as electron acceptor of other microorganism in phototroph-heterotroph interactions [START_REF] Sauze | The interaction of soil phototrophs and fungi with pH and their impact on soil CO2, CO18O and OCS exchange[END_REF], which could be a pathway in PAHs biodegradation. Further studies are required to obtain more information on the metabolism of PAHs in anoxic environments including all the interacting organisms.

I.4.2. Salinity

The effect of salinity on microbial hydrocarbonoclastic activity depends on the variation amplitude of salt concentrations [START_REF] Martins | Biodegradation of petroleum hydrocarbons in hypersaline environments[END_REF]. In some cases, very low concentration of salt reduce hydrocarbon degradation meanwhile optimal biodegradation results in moderate salinity ranges [START_REF] Díaz | Biodegradation of crude oil across a wide range of salinities by an extremely halotolerant bacterial consortium MPD-M, immobilized onto polypropylene fibers: Biodegradation of Crude by Immobilised Bacteria[END_REF]. In contrast, high salt concentrations constitute a stressful agent for most of the known living organisms [START_REF] Kargi | Effect of salt concentration on biological treatment of saline wastewater by fed-batch operation[END_REF][START_REF] Woolard | Biological Treatment of Hypersaline Wastewater by a Biofilm of Halophilic Bacteria[END_REF].

Hypersaline sediments tend to accumulate high amount of organic matter resulting in higher amounts of hydrocarbons entrapped and attached to it (Javor, 1989). Thus, the increment in salinity reduces [START_REF] Brul | Fundibacter jadensis gen. nov., sp. nov., a new slightly halophilic bacterium, isolated from intertidal sediment[END_REF]Gauthier et al., 1992;[START_REF] Le Borgne | Biodegradation of Organic Pollutants by Halophilic Bacteria and Archaea[END_REF][START_REF] Mnif | Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions[END_REF][START_REF] Nicholson | Biodegradation of Benzene by Halophilic and Halotolerant Bacteria under Aerobic Conditions[END_REF][START_REF] Sei | Biodegradation of BTEX at high salinity by an enrichment culture from hypersaline sediments of Rozel Point at Great Salt Lake[END_REF]Yakimov et al., 1998). Among the halophilic fungi, Fusarium lateritium and Drechslera sp. were shown to metabolize crude oil, with degradation efficiency improved more than thrice if petroleum was an additional source of carbon and energy [START_REF] Obuekwe | Growth and hydrocarbon degradation by three desert fungi under conditions of simultaneous temperature and salt stress[END_REF].

Chemically speaking, hydrocarbons are less bioavailable in saline environments than in non-saline. This is the consequence of the "salting out effect" where soluble salts reduce hydrophobic organic compounds solubility in water, the higher the salts concentration in aqueous phase the higher the tendency of organic compounds to be adsorbed in the solid matrix of sediments [START_REF] Means | Influence of salinity upon sediment-water partitioning of aromatic hydrocarbons[END_REF][START_REF] Turner | The influence of salting out on the sorption of neutral organic compounds in estuaries[END_REF]Mackay et al., 2006). It is suggested that PAHs sorption to the sediment particles increases in the presence of salts, not only because the salting out effect on such hydrocarbons but also because of the salting out effect on the organic phase of the sediment, turning entrapped water in sediments, a better solvent for polyaromatics [START_REF] Means | Influence of salinity upon sediment-water partitioning of aromatic hydrocarbons[END_REF][START_REF] Turner | The influence of salting out on the sorption of neutral organic compounds in estuaries[END_REF].

Moreover, high concentrations of salt not only decrease the access to hydrocarbons for microorganisms but alter the concentration of oxygen. In high salinity environments, the oxygen solubility decreases directly proportional with the rise of salts [START_REF] Mcgenity | Halophilic Hydrocarbon Degraders[END_REF][START_REF] Mnif | Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions[END_REF]. This is why, to improve the environmental recovery strategies in marine sediments, an effort should be done to understand the mechanisms of PAHs degradation during anoxic metabolism. A strategy to increase the solubility of hydrophobic compounds used by microorganisms include the production of emulsifying agents which have been reported to be produced by several hydrocarbon-degrading halophiles [START_REF] Al-Mallah | Production of emulsifying agents during growth of a marine Alteromonas in sea water with eicosane as carbon source, a solid hydrocarbon[END_REF][START_REF] Margesin | Potential of halotolerant and halophilic microorganisms for biotechnology[END_REF][START_REF] Martínez-Checa | Yield production, chemical composition, and functional properties of emulsifier H28 synthesized by Halomonas eurihalina strain H-28 in media containing various hydrocarbons[END_REF]Yakimov et al., 1998). Emulsifiers affect the bioabailability of hydrocarbons, which is another of the challenges of marine sediments.

I.4.3. Bioavailability and coefficient of partition

The extent of sorption of non-polar molecules (as PAHs) to the solid matrix has a major influence on its transport and fate in environment. In soil/sediment-water systems, where the organic matter content is significant, the sorption of non-polar molecules occurs largely by partition. This means that one part goes to the solid organic matter and the rest in the water column [START_REF] Chiou | Partition Characteristics of Polycyclic Aromatic Hydrocarbons on Soils and Sediments[END_REF][START_REF] Kile | Partition of Nottpolar Organic Pollutants from Water to Soil and Sediment Organic Matters[END_REF][START_REF] Stuer-Lauridsen | On the influence of the polarity index of organic matter in predicting environmental sorption of chemicals[END_REF], this distribution is called soil/sediment-water distribution coefficient (Kd). The Kd of PAHs is strongly dependent on the organic matter content in soil/sediments. As more organic matter is present in the solid phase, more PAHs are attached to it [START_REF] Kile | Partition of Nottpolar Organic Pollutants from Water to Soil and Sediment Organic Matters[END_REF], creating hydrophobic patches with less bioavailability. The accumulation of PAHs increase as more non-polar molecules and organic matter enter in the environment, thus explain the difficulties in the creation of bioremediation strategies.

I.5. Fungi and bacteria interactions in PAHs degradation

It has been shown that fungi and bacteria collaboration improve degradation of PAHs [START_REF] Li | Biodegradation of aged polycyclic aromatic hydrocarbons (PAHs) by microbial consortia in soil and slurry phases[END_REF].

Mycelia have been found to mobilize entrapped PAHs via vesicle-bound cytoplasmic transport ("hyphal pipelines"; (Furuno et al., 2012) making PAHs bioavailable and thus more amenable for bacterial degradation [START_REF] Fester | Plant-microbe interactions as drivers of ecosystem functions relevant for the biodegradation of organic contaminants[END_REF]Schamfuß et al., 2013) (Figure I.6). Fungal hyphae grow across air-water interfaces, bridge air-filled soil pores, as well as into pore soils with a diameter as little as 2 µm 2 and even penetrate rock matrices (Bornyasz et al., 2005). In addition, fungal mycelia has been shown to be used as paths ("fungal highway" or "fungal translocation networks") for the active (Furuno et al., 2012;[START_REF] Harms | Untapped potential: exploiting fungi in bioremediation of hazardous chemicals[END_REF].

Fungi are known to facilitate bacterial growth, alter bacterial community structures or promote their activity in the hyphosphere by providing sugars and other sources of energy [START_REF] Ingham | Mutually facilitated dispersal between the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex[END_REF][START_REF] Koele | Interactions between mycorrhizal fungi and mycorrhizosphere bacteria during mineral weathering: Budget analysis and bacterial quantification[END_REF]Zhang et al., 2018a). Mycelia also have a great influence on the structure of the surrounding via physical, biological and biochemical processes and often share their microhabitats with bacteria. Bacteria that interact with fungi extracellularly affect fungal development and conidia production. Effects of bacteria on fungal growth, measured by biomass, hyphal branching patterns, elongation, morphology, and density, have been documented (Frey-Klett et al., 2011). Bacteria that colonize the fungal surroundings (hyphosphere) might be essential for the complete depletion of PAHs As an example of the fungal bacterial relations described above, it has been reported that the enzyme laccase sometimes is produced by fungi during interspecific interactions with bacteria [START_REF] White | Extracellular enzyme localization during interspecific fungal interactions[END_REF]. Also laccase activity has been reported to increase when cultures of the white-rot fungi T. versicolor and P. ostreatus are co-cultured with the bacteria E. magnusii, B. subtillis and E. coli [START_REF] Baldrian | Increase of laccase activity during interspecific interactions of white-rot fungi[END_REF]. Since laccase participates in the degradation of some organic pollutants, the result of the interaction between fungi and bacteria demonstrates to be beneficial for PAHs biodegradation, increasing the degradation rate of benzo(a)pyrene in co-cultures compared with single cultures of 1.2 and 3.5-fold order of magnitude [START_REF] Baldrian | Increase of laccase activity during interspecific interactions of white-rot fungi[END_REF]. As a result, the comprehension and seek of fungal and bacterial interactions in PAHs degradation has been carried on, especially on soil microorganisms but is poorly understood in marine environments.

Many fungi detected in marine environments have been already characterized from soil or plant habitats, even when those marine samples were collected from locations far from obvious terrestrial inputs. As previously described, to date the vast majority of fungi identified from marine environments belong to the Ascomycota phylum, also found in soil. Beside this coincidence, fungi specific from sediments have been found indicating that this ecosystem is the reservoir for unexplored fungi and bacteria interactions. Moreover, the capacity of fungal hyphae to transport bacteria in marine ecosystems has not yet been explored. In this habitats, bacterial transport along hyphae find different challenges as water saturation or salt concentration. Particularly, in marine sediments contaminated with oil PAHs accumulate due to their hydrophobicity. This reduces their bioavailability but also create hydrophobic patches that represent physical barriers for bacterial translocation.

We hypothesized that fungal and bacterial interactions are occurring in PAHs contaminated coastal sediments that allows translocation of bacteria through hydrophobic patches. These fungi and bacteria interactions probably involve the selection of bacteria by fungi. In order to gain more information about fungal and bacterial interactions from PAHs contaminated sediments, the aims of this work are i) analyze the impact of PAHs contamination in fungal and bacterial communities interactions in sediments, ii) scrutinize the cultivable diversity of fungi from sediments, their tolerance to PAHs and determine the presence of genes encoding for the extracellular ligninolytic enzymes, and iii) verify if there are fungal selectivity of bacterial communities from sediments under PAHs contamination looking for biomarkers of fungal bacterial interaction. The comprehension and elucidation of fungalbacterial interactions in PAHs contaminated sediments might give important knowledge useful for further implementation of bioremediation strategies to mitigate the effect of PAHs in contaminated environments.

Chapter II. Fungal-bacterial co-occurrence in PAHs contaminated coastal marine sediment: network analysis

II.1. Chapter introduction

Sediments have heterogenic physicochemical characteristics allowing them to be the reservoir of different microbial communities. From these, fungal and bacterial communities are important in the geochemical cycles that regulate sedimentary ecosystems. As expected, these communities share not just space but also relate metabolically creating inter-kingdom interactions. These fungal and bacterial interactions include mutualism antagonism, or synergism. The nature of their dynamic change according to environmental factors. Accordingly, the input of PAHs pollution in sediments, alter the fungal and bacterial interactions shaping microbial community structures. In this chapter, the interaction between fungal and bacterial communities inhabiting PAHs contaminated coastal sediments was evaluated. The interactions were evaluated constructing co-occurrence networks, based on rRNA gene meta-barcoding data, assembled between fungal and bacterial pairs of communities. It is suggested that organisms that co-occur may share similar ecological characteristics [START_REF] Williams | Demonstrating microbial co-occurrence pattern analyses within and between ecosystems[END_REF] making this analysis a tool to elucidate microbial interactions. Furthermore, the effect of PAHs on the diversity of fungal bacterial interactions was also evaluated comparing cooccurrence networks with PAHs contaminated to those from no contaminated samples. This chapter is presented in the format of scientific publication and will be submitted in the journal Environmental

II.2.1. Abstract

Fungal microbiome interacts with the other biotic components in coastal sediment playing a key role in the overall coordination of the whole microbial community. These interactions are affected by human activities, such as the constant affluence of polycyclic aromatic hydrocarbons (PAHs). Although fungi and bacteria interactions have been found to play a key role in PAHs bioremediation in soil, the effect of PAHs on fungal diversity and their specific interactions with bacteria in coastal sediments are yet to be investigated. The understanding of fungal bacterial interactions under PAHs contamination is critical for further bioremediation regarding the important fungal diversity observed in coastal sediment. Here, we investigated the fungal bacterial co-occurrence, and the effect of PAHs in their interactions, in microbial communities from coastal sediments contaminated or not with PAHs. The study confirms the central role that fungi have in coordinating interaction networks with bacteria. The effect of PAHs was obvious on fungi-bacteria network as the abundance and diversity of fungi and bacteria decreased, but the links between the remaining microorganisms were stronger. The network from PAHs contaminated sediments showed that fungi from Chrytridiomycota and Ascomycota phyla have a strong co-occurrence with facultative anaerobic and anaerobic bacteria affiliated to the families Prolixibacteraceae, Fusobacteriaceae and Desulfobulbaceae, not previously described. These results suggest that fungi promote bacterial anaerobic metabolisms, which are important to cope with the presence of PAHs in sediments. Our study reveals the importance of fungal bacterial interactions in coastal sediments paving the way for future studies to fully understand fungal role in coastal sediment.

Keywords: fungal-bacteria interaction, Illumina sequencing, fungal and bacteria diversity, hydrocarbon contamination, co-occurrence network, microbial community

II.2.2. Introduction

Coastal marine sediments are a niche of several interacting microorganisms including algae, archaea, bacteria and fungi. Several studies have shown the crucial role of bacteria and fungi in biogeochemical processes including carbon, nitrogen and sulfur cycling [START_REF] Amend | Fungi in the Marine Environment: Open Questions and Unsolved Problems[END_REF][START_REF] Worden | Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes[END_REF].

In sediment, fungi have shown an active role interacting between them and microbial communities [START_REF] Amend | Fungi in the Marine Environment: Open Questions and Unsolved Problems[END_REF]. Fungal microbiome plays a role in the overall coordination of the whole microbial communities, controlling the structure of sediment microbial functional networks (Booth et al., 2019b). Transcriptomic analyses have demonstrated that fungi and bacteria react to the presence of each other and respond differently depending on the interacting partners [START_REF] Deveau | Pairwise Transcriptomic Analysis of the Interactions Between the Ectomycorrhizal Fungus Laccaria bicolor S238N and Three Beneficial, Neutral and Antagonistic Soil Bacteria[END_REF](Deveau et al., , 2018)).

The presence of polycyclic aromatic hydrocarbons (PAHs) also plays an important role in bacterial community composition and organization [START_REF] Muckian | Bacterial community structure in soils contaminated by polycyclic aromatic hydrocarbons[END_REF]Duran and Cravo-Laureau, 2016).

Because bacteria are important interacting partners with fungi, the changes that occur in bacterial communities have an impact in the complete microbial structure [START_REF] Schulz-Bohm | Fungus-associated bacteriome in charge of their host behavior[END_REF]. Fungi play an important role in cycling anthropogenic carbon sources. Several fungal genera have been linked to PAHs degradation in sediments [START_REF] Walker | Some effects of petroleum on estuarine and marine microorganisms[END_REF] including Cladosporium, Cunninghamella, Penicillium, Fusarium, Alternaria, and Aspergillus [START_REF] Nasrawi | Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico[END_REF][START_REF] Passarini | Marine-derived filamentous fungi and their potential application for polycyclic aromatic hydrocarbon bioremediation[END_REF][START_REF] Steliga | Changes in toxicity during in situ bioremediation of weathered drill wastes contaminated with petroleum hydrocarbons[END_REF] from the phylum Ascomycota (Álvarez- Barragán et al., 2021;Xu et al., 2014;[START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF], some other genera from other phyla as Basidiomycota and Chrytridiomycota.

In PAHs contaminated sediments, fungi are likely primary degraders of high molecular weight hydrocarbons via secreted extracellular enzymes. Fungi work synergistically with PAHs-degrading bacteria (McGenity et al., 2012), fungi and bacteria interactions being beneficial for both partners in PAHs contaminated environments. Mycelia have been found to mobilize entrapped PAHs via vesiclebound cytoplasmic transport (Furuno et al., 2012) making them available for bacterial metabolism. In addition, fungal mycelia are used by bacteria as paths for their active dispersal (Simon et al., 2015), providing sugar and other sources of energy promoting activity in exchange [START_REF] Boer | Living in a fungal world: impact of fungi on soil bacterial niche development[END_REF]. In turn, bacteria also affect fungal development (hypha branching patterns, elongation, morphology and density) as well as their reproduction by stimulating spore generation (Frey-Klett et al., 2011).

As fungal and bacterial interactions are crucial for the functioning of marine sediment environment, the aim of this study is to explore the fungi-bacteria interactions in coastal marine sediments by developing a network approach comparing microbial communities from various geographical locations. We hypothesize that the presence of PAHs contamination will affect the fungi-bacteria interactions resulting in the alteration of network properties. In order to validate the hypothesis, the network constructed form microbial data of PAHs-contaminated sediment was compared with that obtained from un-contaminated sediments.

II.2.3. Material and methods

Coastal sediments from different marine sites were incubated in microcosms mimicking tidal cycles (Stauffert et al., 2013a) or oxygen fluctuations (Terrisse et al., 2017;[START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF], with or without hydrocarbon addition. Sediments were collected and DNA was extracted for microbial characterization. DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio) according to manufacturer's instructions used for later PCR amplifications.

Bacterial 16S rRNA genes were amplified targeting the V4-V5 hypervariable region with the primers 515F (5'-GTGYCAGCMGCCGCGGTA-3') and 928R (5'-CCCCGYCAATTCMTTTRAGT-3') as previously described [START_REF] Wang | Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies[END_REF]. For the identification of fungal communities, the 18S rRNA genes were amplified using the primers FF390 (5'-CGATAACGAACGAGACCT-3') and FR1 (5'-AICCATTCAATGGT-AIT-3') that target the V7-V8 region as described in (Chemidlin [START_REF] Prévost-Bouré | Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR[END_REF]. ( 2021). All PCR mixtures were performed in triplicate in a volume of 25 µL using AmpliTaq Gold 360 Master Mix (Applied Biosystems). The reaction conditions for bacteria amplification include an initial denaturation step at 95°C for 10 minutes, followed by 35 cycles with denaturation step at 95°C for 30 s, hybridization step at 60°C for 30 s, and elongation step at 72°C for 40 s. For fungi, the conditions were: 95°C for 10 minutes, 35 cycles with denaturation step at 95°C for 30 s, hybridization step at 50°C for 45 s, and elongation step at 72°C for 30 s. Sequencing was carried out at the Genotoul platform (Toulouse, France) with Illumina-MiSeq. The 2 x 250 paired-end sequencing gave a full length 411 bp fragment with a 87 bp overlap for the V4-V5 region and a 450 bp fragment with a 150 bp overlap for the V7-V8 region.

To process and delimitate the amplicon sequence variants (ASVs) the open source software Qiime2 (Bolyen et al., 2019) was used for all sequences. The two datasets were first demultiplexed and dada2

was used to control reads quality and infer amplicon sequence variants (ASVs) (Callahan et al., 2017).

Default settings were used except that reverse reads were truncated to 230 base pairs before merging.

The taxonomy was assigned to the ASVs with a fixed sequence similarity threshold of 97% using the SILVA database release 132 for 16S and 18S sequences (Quast et al, 2013). Then, the datasets were filtered to remove mitochondria, chloroplasts and unassigned (at the domain level) sequences.

Phylogenetic trees were constructed using FastTree (Price et al., 2010) in the Qiime2 phylogeny plugin.

The dataset encompassed 66 samples. Both dataset were deposited in the NCBI Sequence Read Archive (SRA) database, the bacterial dataset was assigned with the BioProject ID: PRJNA762061 (SUB10353467). The fungal dataset were assigned with the BioProject ID: PRJNA762024 (SUB10342418).

Co-occurrence network analysis between bacterial and fungal ASVs was inferred from an undirected co-occurrence network. Each network was computed individually using the CoNet (v1.1.1.beta) plugin within Cytoscape (v3.5.1) software [START_REF] Oesper | WordCloud: a Cytoscape plugin to create a visual semantic summary of networks[END_REF]. The detailed use of Cytoscape software can be found at the manufacturer's website (http://manual.cytoscape.org/en/ stable/). In each network, co-occurrence positive interactions were identified using Spearman correlation coefficient > 0. [START_REF] Jeanbille | Response of Core Microbial Consortia to Chronic Hydrocarbon Contaminations in Coastal Sediment Habitats[END_REF].

Co-occurrence between fungi and bacteria has been extensively studied in soils [START_REF] Banerjee | Network analysis reveals functional redundancy and keystone taxa amongst bacterial and fungal communities during organic matter decomposition in an arable soil[END_REF][START_REF] Bell | Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny[END_REF][START_REF] De Menezes | Network analysis reveals that bacteria and fungi form modules that correlate independently with soil parameters: Network analysis of microbial community structure[END_REF] but in sediments, bacterial associations have been considered mainly with eukaryotes [START_REF] Jeanbille | Response of Core Microbial Consortia to Chronic Hydrocarbon Contaminations in Coastal Sediment Habitats[END_REF] overlooking interaction with fungi. Our analysis showed that strong co-occurrences occurs between fungi and bacteria in PAHs contaminated coastal sediments suggesting that fungal and bacterial interactions play a crucial ecological role in contaminated coastal environments.

Modularity analysis revealed a complex structure of 15 modules that represent highly connected ASVs.

Two main modules were identified for including the higher number of nodes and edges (M_1 and M_3,

II.2.4.2. How PAHs contamination affects the microbial community?

In order to evaluate the effect of PAHs contamination in sediment fungal bacterial interactions, two co- In order to analyze the interactions between fungi and bacteria, the relative important members of each network with a Spearman correlation coefficient > 0.82 were identified (Table II.1). The main fungal phyla present in both networks correspond to Ascomycota and Chytridiomycota, which are commonly observed in coastal marine sediment environments (Álvarez-Barragán et al., 2021;[START_REF] Elshafie | Biodegradation of crude oil and n-alkanes by fungi isolated from Oman[END_REF][START_REF] Gleason | Zoosporic true fungi in marine ecosystems: a review[END_REF]. In no contaminated sediments, the ASV Chytridiomycota F876 was found to be associated with cyanobacteria. This was not surprising as Chrytridiomicota are known as parasites of cyanobacteria [START_REF] Rasconi | Use of Calcofluor White for Detection, Identification, and Quantification of Phytoplanktonic Fungal Parasites[END_REF][START_REF] Sønstebø | Possible Implications of Chytrid Parasitism for Population Subdivision in Freshwater Cyanobacteria of the Genus Planktothrix[END_REF]. Moreover, in the nocontaminated network, Chytridiomycota F876 and Chytridiomycota F373 ASVs were associated with sulphate-reducing bacteria (Desulfobacteraceae), which are anaerobic bacteria inhabiting marine sediments [START_REF] Rothermich | Anaerobic, Sulfate-Dependent Degradation of Polycyclic Aromatic Hydrocarbons in Petroleum-Contaminated Harbor Sediment[END_REF]. It is likely that fungi perform the first step in the degradation of complex organic matter [START_REF] Boer | Living in a fungal world: impact of fungi on soil bacterial niche development[END_REF], providing then molecules consumed by sulphate-reducing bacteria, which are usually found in coastal marine sediments playing a key role in the degradation of organic matter [START_REF] Jørgensen | Mineralization of organic matter in the sea bed-the role of sulphate reduction[END_REF].

The network was also composed by one ASV corresponding to the Ascomycota phylum, genus

Penicillium which is commonly found in coastal sediments [START_REF] Amer | Characterization of some fungal strains isolated from the Eastern coast of Alexandria, Egypt, and some applications of Penicillium crustosum. Egypt[END_REF] and was linked with Fusobacteriaceae, Flavobacteriaceae and Rhodobacteraceae bacterial families. For PAHs contaminated sediments network, only the Chytridiomycota ASV F373 was found to be linked with three bacterial families: Desulfobulbaceae, Prolixibacteraceae and Fusobacteriaceae. In comparison with the no contaminated network, the PAHs contaminated network showed less Chytridiomycota members, which is in accordance with previous work reporting that members of the Chytridiomycota phylum are sensitive to hydrocarbon [START_REF] Ventorino | Comparative assessment of autochthonous bacterial and fungal communities and microbial biomarkers of polluted agricultural soils of the Terra dei Fuochi[END_REF] having less resilience capacity compared to other fungi [START_REF] Lemmel | Altered fungal communities in contaminated soils from French industrial brownfields[END_REF]. For example, they produce un-walled spores [START_REF] Longcore | Chytridiomycota. eLS[END_REF] that are more susceptible to environmental changes.

All bacteria associated with fungi in PAHs-contaminated network belong to taxa recognized as anaerobic or facultative anaerobic [START_REF] Ruan | and the study of Actinomycetes systematic in China[END_REF]. Such result suggested that anaerobic metabolism of PAHs has a critical role in structuring the microbial network in contaminated sediment. Anaerobic members have been described within the Chytridiomycota phylum, further supporting the importance of the anaerobic metabolism in microbial assemblage [START_REF] Rezaeian | Methods for the isolation, culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems[END_REF] indicating that fungibacteria interactions are possible under anaerobic conditions. Such result highlights the importance of the Chytridiomycota phylum and their interacting bacteria in PAHs metabolism that have not been yet explored. Noteworthy, most of the ASVs were not affiliated to bacterial genera indicating that most are unknown bacteria, indicating that an effort is required to identify these bacteria associated to fungi. Chapter II. Fungal-bacterial co-occurrence in PAHs contaminated coastal marine sediment: network analysis

II.3. Chapter principal results and discussion

This chapter focused on showing the importance of fungi-bacteria interaction in the microbial community assemblages inhabiting PAHs-contaminated sediments. It has been shown that fungi have a central role coordinating bacterial communities in coastal sediments [START_REF] Roberts | Chytrid fungi shape bacterial communities on model particulate organic matter[END_REF].

Moreover, it has been reported that the microbial communities inhabiting sediments are sensible to PAHs contamination [START_REF] Jeanbille | Response of Core Microbial Consortia to Chronic Hydrocarbon Contaminations in Coastal Sediment Habitats[END_REF]. Thus it is suggested, that the effect of the interaction between fungi and bacteria results beneficial for both partners and allow them to overcome the PAHs pollution hazardous conditions. In our study, co-occurrences between fungi and bacteria in the presence or absence of PAHs were analysed in order to determine the effect of these pollutants on inter-Kingdom interactions. The study is focused on coastal sediments as they have unique characteristics (salinity, oxygen concentration, water saturation, etc.) that in consequence, allow them to have unique fungal and bacterial interacting communities, not yet explored. In this chapter, we showed that fungi have a key role in driving the bacterial community assemblage when exposed to PAHs, fungi ASVs constituting the main nodes linked with several bacterial edges that connected the complete network. Moreover, strong co-occurrence between anaerobic bacteria and the fungal phylum Chytridiomycota was observed, revealing the importance of this fungal phylum recently found abundant in marine environment [START_REF] Gleason | Zoosporic true fungi in marine ecosystems: a review[END_REF]. This observation suggests that marine sediments are the reservoir of unique microbial communities with probably specific interactions. The role that members of the Chytridiomycota phylum play in PAHs degradation has not been yet

described, but the evidence that they are associated with anaerobic bacteria suggests that they promote anaerobic metabolism for PAH degradation. The role of the associated anaerobic bacteria, particularly in PAHs co-metabolism still needs to be elucidated. The results presented here represent a first approach to understand fungal and bacterial interactions in coastal sediments under PAHs contamination. Further analyses are required to obtain more information for a better understanding of the fungi-bacteria interactions in marine coastal ecosystems. In order to bring light on fungalbacteria interaction, fungi from coastal sediments were isolated and characterized for their tolerance toward PAHs (Chapter III). Two of these fungi were then used as translocation networks in fungalbacterial interaction experiments in order to describe the bacterial communities interacting with fungi in presence of PAHs (Chapter IV).

Chapter III. Fungi in PAH-contaminated marine sediments: Cultivable diversity and tolerance capacity towards PAH

III.1. Introduction

In the previous chapter (Chapter II), a co-occurrence network analysis was performed to evaluate the interactions with fungi and bacteria communities from PAHs contaminated coastal sediments, and the impact that PAHs pollution have in these interactions. The results of these analyses showed the role that fungi have on coastal sediments coordinating the structure of microbial communities and the relevance of fungi and bacteria interactions toward PAHs pollution. Considering the importance that fungi have over the population of bacterial communities and the possible metabolism of PAHs, this chapter (Chapter III) is dedicated to the isolation of fungi from coastal sediments that will enable to further evaluate fungal bacterial interactions (Chapter IV). In order to have a wide view of the cultivable fungal diversity inhabiting coastal sediments, 85 fungi were isolated on seawater media contaminated with PAHs. The fungal isolates were then identified using the ribosomal internal transcribed spacer (ITS), which allows the evaluation of cultivable diversity belonging to these phyla, including the possibility of new species. Following this, the fungi were tested for their ability to tolerate and remove four PAHs from the media (phenanthrene, pyrene and fluoranthene, and benzo[a]pyrene)

with gas chromatography technique. Furthermore, a scrutiny of the genes that encode enzymes related to PAHs extracellular degradation was performed searching for manganese peroxidase, lignin peroxidase and laccase genes. Considering the importance for fungal bacterial interactions the capacity that fungi have to internalize PAHs, two fungi were selected and the intracellular uptake and transport of pyrene along the hyphae was analysed by microscopic observations.

Microscopic analyses presented in this work

III.2. Fungi in PAH-contaminated marine sediments: Cultivable diversity and tolerance capacity towards PAH

Chapter III. Fungi in PAH-contaminated marine sediments: Cultivable diversity and tolerance capacity towards PAH

III.3. Chapter principal results and discussion

As seen in Chapter II, fungi and bacteria have networks of interactions in PAHs polluted coastal sediments. In order to further analyse these interactions, 85 fungi from coastal sediments were isolated in seawater media to keep salinity conditions. The identification, based on the ITS gene sequence, revealed that 83 (97.6%) isolates were members of the phylum Ascomycota and 2 (2.4%) of the phylum Basidiomycota. Among the 85 fungi, 48% of the isolated strains exhibited PAHtolerance with a removal capacity above 60%. Such results demonstrate that sediments are an important reservoir of PAH -tolerant fungi that has not yet been completely explored. The capacity of fungi to tolerate PAHs was not correlated with the presence of ligninolytic enzymes genes that are related to extracellular degradation of PAHs, suggesting other strategies for PAHs removal, such as intracellular degradation via cytochrome P450 or PAHs storage. Surprisingly, the storage of PAHs was also observed to occur differently between fungi, the fungus with the highest removal rates (Alternaria destruens F10.81) store PAHs in the cytoplasm while the fungus with the lowest removal rates (Fusarium pseudonygamai F5.76) store PAHs in vacuoles. Further analysis must be done to determine whether these strategies are related to removal capacities or to different PAHs metabolisms. The fungal uptake of PAHs is important not only for decreasing pollutant content in the environment (decreasing environmental impact), but also for the translocation of PAHs via the fungal hyphae resulting in PAHs bioavailable for degrading bacteria in other locations (Furuno et al., 2012).

Moreover, it is known that fungi transport not just PAHs but also bacteria that travel along the zone surrounding the hyphae (the "hyphosphere") for distant locations (Banitz et al., 2013). The potential of the two selected fungi (Alternaria destruens F10.81 and Fusarium pseudoygamai F5.76) to translocate single bacteria and the possible selection of bacteria within a microbial community by fungi is evaluated in the following chapter (Chapter IV).

Chapter IV: Marine fungal translocation network transport aerobic/anaerobic bacteria from PAHs contaminated sediments

IV.1. Chapter introduction

Considering the uptake of PAHs by fungi as a factor that aid bacteria in PAHs degradation (Banitz et al., 2013), two fungi (Alternaria destruens F10.81 and Fusarium pseudoygamai F5.76) were chosen for their high and low PAHs removal capacity (described in Chapter III). Furthermore, studies in soil have demonstrate that fungal hyphae can be used by PAHs tolerant bacteria to translocate from one site to another (see "fungal highway" in Chapter I), allowing bacteria to reach unless unaccessible locations (Kohlmeier et al., 2005). As sediments are water-filled environments, PAHs accumulate in hydrophobic patches, which are less accessible to reach by for PAHs degrading bacteria. For this reason, in this chapter (Chapter IV) is evaluated the capacity of fungal hyphae to be used as translocation networks by bacteria through PAHs contaminated sediments (mimicking PAHs polluted environment), which

has not yet been described.

IV.2. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are of major concern for the environment because they are toxic and resilient due their hydrophobic nature (Duran and Cravo-Laureau, 2016). They tend to bind to particles in soil and sediments (Sanglard et al., 1986), creating patches heterogeneously distributed and decreasing their bioavailability (Worrich et al., 2016). Microorganisms and their interactions play a key role in PAHs degradation (Duran and Cravo-Laureau, 2016;McGenity et al., 2012). Among these interactions, fungal bacterial interactions (FBI) have been demonstrated to stimulate PAHs degradation (Worrich et al., 2016).

Bacteria co-exist with fungi as free living organisms, or in the fungal intracellular environment (obligate endobacteria) or both (facultative endobacteria) (Olsson et al., 2017). The number of bacteria able to stablish symbiotic interactions with fungi seems to be limited (Baschien et al., 2009;Lyons et al., 2005;Olsson et al., 2017;Scheublin et al., 2010;Simon et al., 2015;Warmink et al., 2009) indicating a selectivity in FBI. Bacteria associate directly with the fungal hyphae (Baschien et al., 2009;Cuong et al., 2011) living in the hyphosphere, the volume influenced by the hyphae (Olsson et al., 2017). Bacteria colonizing the hyphosphere are essential for the depletion of PAHs (Harms and Wick, 2006;Kohlmeier et al., 2005) The FBI in the hyphosphere has been demonstrated to be synergetic and mutualistic where both partners get benefit (Olsson et al., 2017). Bacteria influence the metabolism and physiology of fungi (Barron, 2003;Frey-Klett et al., 2011;Netzker et al., 2015), while fungal hyphae transfer nutrients, water and pollutants to bacteria (Banitz et al., 2013;Furuno et al., 2012;Leveau and Preston, 2008;Wick et al., 2007;Worrich et al., 2017). In addition, fungal mycelia has been demonstrated to be used as translocation networks (fungal highways) for the dispersion of bacteria (Abeysinghe et al., 2020;Kohlmeier et al., 2005;Simon et al., 2015), particularly in soil allowing bacteria to colonize different niches and crossing obstacles (Bornyasz et al., 2005;Worrich et al., 2016). The bacteria dispersion via mycelia has been shown to stimulate PAHs degradation when pollutants or bacteria are heterogeneously distributed (Kohlmeier et al., 2005;Worrich et al., 2016). Translocation networks have been largely studied in soils but no attention has been paid on translocation networks in marine sediments because it is assumed that the translocation of bacteria is not limited in aquatic environments (Grossart et al., 2001). Nevertheless, bacteria have to face different obstacles for dispersion in aquatic environments including hydrophobic patches formed by hydrocarbons (Fu et al., 1994), plastics (Biermann et al., 2020) and organic matter aggregates (Maggi and Tang, 2015). It has been demonstrated that fungi extend their mycelia through marine sediments providing space and support for bacteria (Booth et al., 2019). In marine ecosystems a large diversity of fungi has been described, the vast majority of fungi belonging to the Ascomycota and Basidiomycota phyla (Álvarez-Barragán et al., 2021;Xu et al., 2014;[START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF]. We hypothesized that to overcome obstacles, bacteria can use fungi as translocation networks in marine environments through FBI probably selecting bacteria. We setup a specific experimental design for testing i) the capacity of bacteria to use fungal translocation networks to overcome patches formed by PAHs contaminated sediments, ii) the specificity and selectivity of the FBI.

IV.3. Material and methods

IV.3.1. Media and sediments

To mimic marine conditions, seawater minimal media (swMM) was used with the following composition: KCl 0.75 g/L, CaCl•2H2O 1.47 g/L, NH4Cl 1.

IV.3.2. Fungal selection

Two fungi of high and low PAH-removal capacity (A. destruens F10.81 and F. pseudonygamai F5.76 respectively) were isolated from a heavily PAH contaminated coastal sediment (Etang de Berre, France) previously described (Álvarez-Barragán et al., 2021). Fungi were mantained in MDA (malt dextrose agar) elaborated with swMM to keep salinity conditions (MDAsw).

IV.3.3. Bacteria isolation

Sediments from Etang de Berre were inoculated directly in LB media prepared with swMM (LBsw) to keep salinity. After 48h of growht, eight bacteria with different phenotipic colony formation were selected and reinoculated in separated plates of LBsw media until pure cultures were obtained.

Bacteria were evaluated for swimming and swarming motility and their capacity of growth in 30 mgL-1 of pyrene as only carbon source. DNA from bacteria was extracted from isolated colonies in swLB media using the QUIAGEN DNeasy® UltraClean® Microbial Kit (Cat. No. 12224-40) following the manufacturer instructions. Bacteria were identified by the 16S gene amplifying a consensus sequence with the forward primer 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and reverse primer 1387r (5′-GGG CGG WGT GTA CAA GGC-3′) with an approximate length of 1,300 bp (Marchesi et al., 1998). 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.

IV.3.4. Experimental system

Bioessays were assesd by placing in Petri dishes (5 of diameter) fulled with swMM containing 10% of swLB media and 30 mgL-1 of pyrene, letting separation gaps of 2 mm that create two proportions: 1.4 and 3.4 cm (Figure 1A-a). The gaps were filled either with sterile wet sand, sterile sediment, wax or let empty (emulating air pores) to mimic spatially distinct barriers as in heterogenic contaminated sediment (Figure 1B). The barriers were chosen due to the differences of hydrophobic and hydrophilic characteristics.

Actively growing mycelia was picked up from PDA dishes and inoculated at 3 mm from the border in the bigger side and it was let growh until the hypha grow 1 cm long (48 hours) (Figure 1A-b). Bacteria (pregrown for 24 h in liquid swLB cointaining 30mgL-1 pyrene) were inoculated close the micelia growht (Figure 1A-c). The setups were incubated in darkness for 10 days until the mycelia growht beyond the barriers (Figure 1A-d). Bacterial translocation along hyphae was assessed by microscopic observation. All experiments were carried out in triplicate.

IV.3.5. Bacterial communities transported by translocation networks

The same setups previously descrived were used with F. pseudonygamai F5.76 and A. destruens F10.81 and glass fibbers (abiotic control) as translocation networks and a sediment gap was placed. To evaluate the transport and selection of bacterial communities along the translocation networks, sediments from Etang the Berre or Canal Vieil were used as inoculums. The experimental system was as previously described. The sampling was performed at four points: in the inoculum, after the inoculum, in the gap and after the gap (Figure 1C). Fungal growht and bacterial colonies were observed after incubation time (Figure 1D) and all samples were collected at the same moment and placed in independent Eppendroff tubes. To asses the cultuvable bacterial community in the media used, sediment from both sources was inoculated in Petri dishes without translocation network and recovered in the same collection times. All samples were performed in triplicate. with fused MiSeq adapters and heterogeneity spacers in a 25-μl PCR. The hypervariable region of the 16S rRNA genes were performed using the bacteria primers 344F-V3V4 (5'-ACGGRAGGCAGCAG-3') and 801R-V3V4 (5'-TACCAGGGTATCTAATCCT-3'). The reverse and forward primers included Illumina adapters. Before amplification, the concentration of DNA was determined with a Quant-It kit in microplate in order to normalize the following amplification. Amplifications were performed in triplicate using AmpliTaq Gold 360 Master Mix (Applied Biosystems) to prepare 25 µL reactions containing 2 ng of DNA of Bacteria. Cycling conditions were: an initial denaturation step at 95°C for 10 minutes, followed by 35 cycles with denaturation step at 95°C for 30 s, hybridization step at 60°C for 30 s, and elongation step at 72°C for 40 s or 30 s, after which a final elongation step at 72°C for 10 minutes was performed. Sequencing by synthesis was carried out at the Genotoul platform (Toulouse, France) with Illumina-MiSeq. The 2x 250 paired-end sequencing gave a full length ~460 bp for the V3-V4 region.

IV

IV.3.7. Sequence processing and ASV delimitation

The open source software Qiime2 (Bolyen et al., 2019) was used for 16S. Sequencing resulted in 2985157 raw reads. The dataset was first demultiplexed and dada2 (Callahan et al., 2016) was used to control reads quality and infer amplicon sequence variants (ASVs) (Callahan et al., 2017). Default settings were used except that reverse reads were truncated to 230 base pairs before merging. This denoising step resulted in a 1699056 trimed sequences dataset. The taxonomy was assigned to the ASVs with a fixed sequence similarity threshold of 97% using the SILVA database (Quast et al., 2012) release 132 for 16S sequences. Then, the dataset was mitochondria, chloroplasts and unassigned (at the domain level) filtered. Bioinformatics treatment resulted in 1882 prokaryotic ASVs. Phylogenetic trees were constructed using FastTree (Price et al., 2010) in the Qiime2 phylogeny plugin. The phylogenetic tree was made ultrametric for the following analyses using the "chronos" function in the R package ape (Paradis et al., 2004) with branch lengths representing relative time and all tips equidistant from the root. The dataset encompassed 84 samples. The complete dataset was deposited in the NCBI Sequence Read Archive (SRA) database and is available under the BioProject ID PRJNA761592 (Submission ID: SUB10342502).

IV.3.8. Statistics

Statistical analysis of ASV richness of each sample and diversity index Shannon were calculated comparing translocation networks and sampling points in R software version 4.0.3 (http://www.rproject.org/). Unconstrained non-metric multidimensional scaling (NMDS) for sampling points and translocation networks was performed using Bray-Curtis distances in the metaMDS function (k=3) of the vegan package 2.5-6. Differences of translocation network effects on the microbial communities were tested using permutational multivariance analysis of variance (PERMANOVA) with 9999 permutations. Statistical differences in relative abundance of phylums between same groups were using two sample homoscedastic t-test, two-taile. To identify the significant abundant bacterial ASVs from both sediments between translocation networks after the gap, finear discriminant analysis effect size (LEfSe) analysis was applied, the analysis was run in Hutlab Galaxy (https://huttenhower.sph.harvard.edu/galaxy/).

IV.3.9. In vivo mapping of oxygen concentration in the mycopshere:

A plannar optode sensor foil (SF-RPSu4, PreSens, Regensburg, Germany) and a VisiSens TD Detector Unit DU02 (PreSens, Germany) were used for time-lapse optical mapping of oxygen concentration in the mycopshere of A. destruens F10.81 and F. pseudonygamai F5.76 (cf. SI and Figure 5A for the calibration procedure of the sensor foils and calibration data). Shortly, a sensor foil (1 cm × 1 cm) was glued to the bottom of a Petri dish (μ-Dish 35 mm, low, Ibidi, Gräfelfing, Germany) using silicon glue.

The Petri dish was kept in the dark to let the silicon glue cure overnight. After that a circular agarose pad (Ø: 18 mm; h: 0.7 mm, swMM + 10% of swLB media + 30 mg L -1 of pyrene) was prepared as described by Young et al. (2012) and placed on top of the sensor foil. A circular agarose pad (Ø: 10 mm) with the fungal inoculum was then placed at 1 mm distance from the oxygen sensing pad. An observation area (1.8 × 2.5 mm, 4 mm from the fungal inoculum) was chosen in the oxygen monitoring pad to monitor oxygen dynamics during fungal habitat colonization. Specifically, images of the observation area were taken by the detector unit (exposure 100000 µm, gain 10) with a 1 h imaging interval during the 96-hour incubation. Time-lapse oxygen mapping experiments were performed in triplicate each for A. destruens F10.81 and F. pseudonygamai F5.76. Another, identical setup was used to map the spatial oxygen and mycelial distribution of fungus A. destruens F10.81 after 48 h incubation of the whole oxygen monitoring area (1 cm × 1 cm). To screen for oxygen in the 1 cm x 1 cm sensor foil, 30 spots (cf. 1.8 × 2.5 mm per spot) were imaged by the VisiSens TD Detector Unit as follows: the position of VisiSens TD Detector Unit was fixed while the position of the Petri dish was precisely shifted using a Nikon microscope stage (Nikon AZ100, Tokyo, Japan) controled by the microscope imaging softerware (large image accuisition, NIS-ELEMENTS, Basic Research, Nikon). After that, microscopic images were immediately taken to visualize mycelial distribution in the direct vicinity of the sensor foil using the Nikon microscope (AZ100, Tokyo, Japan). Briefly, bright-field images were taken by the Nikon micrscope with 5× objective and 3× microsope magnification (a total magnification of 15 ×, 1.78 × 2.22 mm per image) with LED illumination (exposure time 30 ms). Images taken by the VisiSens TD Detector Unit (spatial O2 information) and Nikon microscope (mycelial distribution) were grouped separately, and then the size of the two grouped images were stadardized to 1.87 µm per pixel using ImageJ. The standardized images were then overlaied in ImageJ with 50% opacity to combine the spatial oxygen information and the mycelial distribution in the microcosm.

IV.4. Results and discussion

IV.4.1. Bacterial requirements for translocation in fungal networks

In order to determine the needed characteristics of bacteria able to use the fungal highway as translocation network, eight bacteria isolated from coastal sediments were tested (Table IV.1). Seven bacteria were able to tolerate the presence of pyrene, while the bacterium B2 sensitive to pyrene, served as control for PAHs growth capacity (Table IV.1). All bacteria were motile (swimming or swarming) except the bacterium B3 selected as control for motility (Table IV.1). IV.1). These results showed that motility is a crucial characteristic of bacteria for transport along the translocation networks, as previously reported for isolated bacteria (Kohlmeier et al., 2005).

The mycelia of

But information with complex bacterial communities is still scarce [START_REF] Zhang | Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes[END_REF]. Although bacteria were transported through the gaps irrespective of the hydrophobicity, only three bacteria were transported through the air gap. Such observation was surprising since several studies reported the capacity of motile bacteria to cross air gaps through fungal translocation networks (Furuno et al., 2012;Kohlmeier et al., 2005;Simon et al., 2015). It is likely that fungal networks require a physical support for bacterial translocation (Table IV.1). Indeed, motile bacteria were translocated through hydrophilic and hydrophobic gaps in both fungal translocation networks, which may be explained by the capacity of fungi to modify their surface hydrophobicity (Unestam and Sun, 1995). The main difference between A. destruens F10.81 and F. pseudonygamai F5.76 translocation networks relies in the capacity of bacterium B8 to cross hydrophobic gaps with F. pseudonygamai F5.76 whereas it crosses the hydrophilic wet sand gap with A. destruens F10.81. The mechanism modifying fungal surface involves the production of various hydrophobins (Wösten et al., 1994) probably explaining the differences observed between fungal translocation networks. As A. destruens F10.81 and F. pseudonygamai F5.76 were able to translocate bacteria, they were used to study translocation of microbial communities.

IV.4.2. Bacterial selection by fungal translocation network

Specific experimental design was setup allowing the analysis of bacterial transport from the inoculum and at different sampling points through translocation networks crossing a sediment gap (Figure 1C).

Two different sediments (Canal Vieil and Etang de Berre) were selected as inoculum in order to determine the specificity of the fungi-bacteria interaction. The bacterial communities collected on the sampling points on the fungal translocation networks (A. destruens F10.81 and F. pseudonygamai F5.76) were compared to those obtained with a glass fibber translocation network control. The sequencing of the bacterial communities provided 2985157 reads of high quality sequences, after trimming the obtained 1699056 sequences were distributed within 1882 ASVs (Table IV.2).

For the Canal Vieil sediment, the inoculum showed similar Shannon index (H) irrespective of the presence or not of a translocation network (Table IV.2). In contrast, for Etang de Berre sediment, the Shannon index (H) of the inoculum was significantly higher (t-test, p-value < 0.05) for F. pseudonygamai F5.76, lower for glass fibbers (t-test, p-value < 0.05), and similar for A. destruens F10.81. Thus, the presence of translocation networks had different effect according to the origin of the microbial community. Indeed previous studies have shown that the microbial community composition is dependent on physical-chemical parameters (Stauffert et al., 2013), which probably affect the bacterial population able to be translocated. For A. destruens F10.81 translocation network, the Shannon index (H) was significantly different (t-test, p-value < 0.05) along the sampling points only for Etang de Berre sediment (Table 2). For F. pseudonygamai F5.76 translocation network, the Shannon index (H) was significantly lower (t-test, p-value < 0.05) in the gap and after the gap compared to the inoculum. For glass fibbers translocation network, the Shannon index (H) was significantly lower (ttest, p-value < 0.05) only for after the gap with Etang de Berre sediment. Thus, the nature of the translocation network affect the diversity of microbial communities along the sampling points suggesting a selection of bacterial communities related to fungi-bacteria interaction. Consistently, several studies have demonstrated fungal-bacterial interactions (FBI) involving complex molecular mechanisms (Deveau et al., 2018;Schmidt et al., 2016), which might results on bacterial community selection [START_REF] Zhang | Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes[END_REF]. In order to characterize the FBI and demonstrate bacterial selection, the structure of the bacterial community was analysed. Richness (ASV) 260.67 ± 76.22 116 ± 43.49 102.33 ± 14.04 26 ± 15.87 31.33 ± 8.5 35 ± 20.42 81.67 ± 51.29 41.33 ± 4.51 43.67 ± 35.8 17.67 ± 8.96 114.67 ± 23.12 48.67 ± 13.5 41.67 ± 8.96 42.33 ± 24.21 Shannon (H) Observed 323 ± 40.15 110.33 ± 54.63 109 ± 25.24 28.67 ± 9.61 6.67 ± 5.51 25.67 ± 22.18 129 ± 12.29 64.67 ± 72.28 27.67 ± 4.51 36.67 ± 37.54 74.33 ± 11.37 33.33 ± 14.47 52.67 ± 50.58 38.33 ± 25.11 Shannon (H) Analysis were performed in triplicate; 1 Cultured along 5 days at 25 °C in sea water minimal media added with 10% LB and 30 mg/L of Pyrene; 2 Indicate sampling points in the fungal translocation network setup.

The bacterial communities of the inocula from both Canal Vieil and Etang de Berre sediments were different from those collected along the sampling points (before the gap, in the gap and after the gap) showing that fungal networks shape bacterial community structures [START_REF] Zhang | Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes[END_REF]. sediments. This observation suggested that the presence of fungi create specific conditions favoring the growth of such microorganisms under the experimental conditions. It is important to note that most of these dominant genera are known to be motile (van Belkum, 2006), which is a feature that has been shown to be crucial for hyphosphaere colonization [START_REF] Zhang | Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes[END_REF]. In addition, these genera have probably the capacity to resist to PAHs and grow fast in the used cultured media facilitating the colonization. Indeed, Amphritea, Arcobacter, Celeribacter, Marinifilum, Marinomonas, Oceanospirillum, Pseudoalteromonas, Spirochaeta, and Vibrio are known to adopt a K-strategy, fast growing in presence of hydrocarbons (Chronopoulou et al., 2015;Hedlund and Staley, 2001;Lai et al., 2014;Miller et al., 2019;[START_REF] Nuñal | Hydrocarbon Degradation and Bacterial Community Responses During Remediation of Sediment Artificially Contaminated with Heavy Oil[END_REF]Terrisse et al., 2017;Xiong et al., 2015).

The bacterial composition was modified in the sampling points (before, in the gap and after) along the translocation networks in agreement with NMDS. As a result, Pseudoalteromonas was found dominant after the gap for all translocation networks. Obviously, Pseudoalteromonas was the most adapted to the experimental conditions allowing it to fully cover the hyphosphere. Nevertheless, different genera

IV.4.3. Specific association between bacteria and fungi

In order to identify the ASVs specifically associated to a translocation network the LEfSe analysis was performed considering both sediments after the gap. LEfSe revealed statistically significant more abundant ASVs for a condition that have been proposed as a biomarker (Segata et al., 2011). the presence of hydrocarbons (Hedlund and Staley, 2001;Llobet-Brossa et al., 1998;Paissé et al., 2008;Ye et al., 2019;[START_REF] Zhao | Thalassospira xianhensis sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium[END_REF], and to the best of our knowledge they have been not described interacting with Alternaria sp. The selection of bacteria by fungi has been described to relies on the hyphal production of compounds with either chemio-attractive effect (Deveau et al., 2010) or promoting bacterial growth (Bravo et al., 2013), while fungi receives vitamins from bacteria (Abeysinghe et al., 2020). It was surprising that Spirochaeta litoralis was identified as a biomarker for translocation network under our experimental aerobic conditions because it is known as strict anaerobic bacteria (Hespell and Canale-Parola, 1970;Subhash and Lee, 2017). Such observation suggested that anaerobic microniches were created along the fungal translocation network development.

IV.5. Conclusion

Despite the fact that our experimental conditions selected the fast growing bacteria Pseudoalteromonas, our study demonstrated that the fungal translocation network selected specific bacterial populations, especially for A. destruens F10.81 while no specific biomarker was identified for F. pseudonygamai F5.76. For A. destruens F10.81 four high specific biomarkers were identified. Among these biomarkers, the most tricky was the observation of anaerobic bacteria (Spirochaeta litoralis). [START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF]. Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic). Sci. Rep. 5.

Zhang, Y., Kastman, E.K., Guasto, J.S., and Wolfe, B.E. (2018). Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes. Nat. Commun. 9. [START_REF] Zhao | Thalassospira xianhensis sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium[END_REF]. Thalassospira xianhensis sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium. Int. J. Syst. Evol. Microbiol. 60, 1125-1129. Chapter IV. Marine fungal translocation network transport aerobic/anaerobic bacteria from PAHs contaminated sediments

IV.3. Chapter principal results and discussion

In the previous chapter (Chapter III), two fungi were isolated and selected for their low and high PAHs removal capacities (Fusarium pseudonygamai F5.76 and Alternaria destruens F10.81, respectively).

These fungi were consequently used to analyze bacterial transport and selectivity along the fungal mycelia considering the challenges that PAHs contaminated coastal sediments imply. First, the transport via fungal translocation networks of eight bacteria isolates was tested across different gaps (wax, sediment, wet sand and air) mimicking the hydrophobic patches that PAH contamination create in sediments. Such analysis showed that bacteria able to cross the gaps among fungal hyphae needed to be motile and tolerant to PAHs contamination. Furthermore, the selection of bacterial communities according to different fungal translocation networks was assed and evaluated. The bacterial communities obtained from all translocation networks showed mainly dominance of the fast growing bacteria Pseudoalteromonas for all networks. Nevertheless, each translocation network has its own bacterial populations demonstrating fungal selectivity for specific bacteria members. Then, deeper analysis of the bacterial communities allowed to associate four bacterial biomarkers to the fungus A. destruens F10.81 belonging to Thalassospira sp., Spirochaeta litoralis, Arcobacter sp. and Vibrio sp. So far, these bacteria have not been reported to be associated to fungal partners, nevertheless all the bacteria mentioned have been reported to be associated to PAHs degradation and marine environment. For this reason, further efforts should be done to understand the role of fungal and bacterial interactions in PAHs contaminated sediments.

Finally, in Chapter II, the co-occurrence between fungi and anaerobic bacteria was shown, suggesting that fungi-bacteria interactions are important in the organization of microbial communities.

Consecutively in this Chapter IV, we present the detection of the anaerobic bacteria Spirochaeta litoralis as specific biomarker of A. destruens F10.81 that supports our previous finding. Coupled with this, measurements of the concentration of oxygen around the hypha during its development, confirmed the formation of oxic and anoxic zones that allow the propagation of aerobic and anaerobic bacteria along the fungal translocation network. The results previously described demonstrate that coastal sediments are a source of unexplored interactions and open new questions about fungal and bacterial strategies to overcome PAHs polluted environments, which will be discussed in the Perspectives presented in Chapter V.

Chapter V. Conclusions and perspectives

V.1. Conclusions

Based in the current knowledge of fungal and bacterial interactions under PAHs contamination exposed in Chapter I, with this work we do an effort to understand the fungal and bacterial interactions from coastal sediments considering the particular characteristics of these ecosystems. Supported by chemical, microbiological and molecular techniques, it was possible to make an analysis of the impact of PAHs in the fungal and bacterial diversity dynamics. The analysis present first, the role of fungi as key organisms able create networks of interactions with bacterial communities under PAHs contaminated coastal sediments (Chapter II), suggesting they play a key role in microorganisms dynamics. The diversity of interactions between fungi and bacteria were proved to decrease in number but increase in strength under PAHs pollution, demonstrating the importance of fungal and bacteria co-occurrence for the microbial resilience against pollutants in these ecosystems (Chapter II).

Additionally, this analysis showed for the first time the association of anaerobic bacterial communities with facultative anaerobic fungi from the Chytridiomycota phyla. Such result shine light the need of further exploration and understanding of coastal sediments as source of undescribed fungal and bacterial interactions.

In order to understand the role and impact of fungi in PAHs contaminated coastal sediments and to allow us to select two fungi as research models (under laboratory conditions), the cultivable diversity of fungi from coastal sediments was isolated and identified. The fungal diversity was found to be rich in fungi from the phylum Ascomycota (Chapter III) revealing a reservoir of undescribed fungi, calling the attention for further research of new fungal taxa in these ecosystems. Different fungi from coastal sediments result in different degrees of PAHs removal capacities. Considering the toxic effect of PAHs for organisms [START_REF] Haritash | Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review[END_REF], the removal of PAHs from the ecosystem might help buffering the environmental impact of these pollutants. Interestingly, the microscopic analysis presented in Chapter III showed two different strategies of removal and storage of PAHs that include cytoplasmatic reserve and vacuole packaging. Further analysis must be performed to know if these different mechanisms affect PAHs degradation and their impact on the fungal metabolism of PAHs molecules. Moreover, the PAHs depletion showed to be independent of the genes of extracellular peroxidases and neither to the capacity of fungi to grow in contaminated media, demonstrating the elasticity of fungal metabolism, which allow them to be highly tolerant to pollutants. To see the impact that different uptake strategies and removal of PAHs might have in the possible associated bacterial communities, two fungi were chosen (Alternaria destruens F10.81 and Fusarium pseudoygamai F5.76) for studying the fungi-bacteria interactions.

Profiting the capacity of fungi to translocate bacteria from one site to another (Kohlmeier et al., 2005), in Chapter IV the analysis of bacterial communities associated to the two fungal translocation networks chosen in Chapter III is presented. Bacterial communities showed different bacterial translocation characteristics through heterogeneous patches suggesting that fungal translocation might take place in PAHs contaminated coastal sediments. Moreover, different fungal networks shape different bacterial communities during the translocation demonstrating selectivity. Specificity of bacterial translocation through fungal hypha was also demonstrated with the finding of bacterial biomarkers associated to one of the fungus, as a result of the interaction. This work gives the first approach to study fungal and bacterial specific interactions and transport in coastal sediments, but more analysis should be done to understand how these interactions occur. The most surprising result was the transport of anaerobic bacteria described for the first time so far. Analysis of the hyphosphere concentration of oxygen showed that fungal metabolism create niches for aerobic and anaerobic bacteria depending on the hypha stage of development. This information support the previous findings of anaerobic bacteria and facultative anaerobic fungi that co-occur in PAHs contaminated sediments showed in Chapter II.

V.2. Perspectives

The results presented in this work constitute the first basis for understanding fungal-bacterial interaction in PAHs contaminated coastal sediments. The role of fungi as key taxa in coordinating bacterial communities assemblage of selected bacteria was observed in PAHs polluted media.

However, it remains unknown whether the selection of bacterial communities from fungal mycelia favours the uptake and degradation of PAHs. Thus, further analyses are required to characterize PAHs removal in fungi-bacteria co-cultures (Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76 associated with the bacterial communities obtained after the gap). The co-culture analysis will bring light on the capacity of the bacterial communities, selected by fungal mycelia, to aid in the decontamination of PAHs polluted environments. The analysis will be also helpful to describe the possible synergism between fungi and bacteria under hazardous conditions. Such information will provide new insights on the behaviour of fungi-bacteria interactions in coastal sediments, useful information to understand the strategies that these microorganisms use to overcome pollutants as PAHs.

In order to have molecular insight of fungal-bacterial interactions, a molecular approach targeting volatile organic compounds (VOC) that have been reported as drivers of the interactions between fungi and bacteria (Deveau et al., 2018), will allow to understand the communication between fungal and bacterial cells. Transcriptomic analyses have demonstrated that fungi and bacteria react each other, responding differentially according to the interacting partners. Several molecules may be used by microorganisms for mutual detection, most of them being based on small signaling molecules [START_REF] Scherlach | Molecular Bacteria-Fungi Interactions: Effects on Environment, Food, and Medicine[END_REF]. Also, it has been reported that signaling molecules modify the metabolism of the interacting partner, resulting in growth promotion, enzyme production and enhanced reproduction [START_REF] Crowe | Induction of Laccase Activity in Rhizoctonia solani by Antagonistic Pseudomonas fluorescens Strains and a Range of Chemical Treatments[END_REF][START_REF] Fiddaman | The production of antifungal volatiles by Bacillus subtilis[END_REF][START_REF] Zhao | Antifungal, Insecticidal and Herbicidal Properties of Volatile Components from Paenibacillus polymyxa Strain BMP-11[END_REF]. For example, Pseudomonas fluorescens has been shown to induce the production of laccase in Rhizoctonia solani in co-cultures mediated by VOCs [START_REF] Crowe | Induction of Laccase Activity in Rhizoctonia solani by Antagonistic Pseudomonas fluorescens Strains and a Range of Chemical Treatments[END_REF]. The whole VOCs pattern in intra-kingdom is still not elucidated. Moreover, considering the findings of fungal and bacteria anaerobic interactions presented in this thesis, further efforts should be done to elucidate how the presence of PAHs enhance the anaerobic cometabolism of these pollutants. Studies related to the anaerobic PAHs degradation of fungal-bacterial consortium have been proposed [START_REF] Elyamine | Aerobic and Anaerobic Bacterial and Fungal Degradation of Pyrene: Mechanism Pathway Including Biochemical Reaction and Catabolic Genes[END_REF] but not yet realized. The anaerobic PAHs degradation by fungi has recently gain interest as more evidence emerges, new fungal members are classified [START_REF] Amend | Fungi in the Marine Environment: Open Questions and Unsolved Problems[END_REF] and the PAHs metabolic pathways are elucidated [START_REF] Aydin | Aerobic and anaerobic fungal metabolism and Omics insights for increasing polycyclic aromatic hydrocarbons biodegradation[END_REF].

It is important to highlight that sediments are highly heterogeneous from a location to another, making the possible fungal and bacterial interactions also diverse. As presented in Chapter II, a co-occurrence network approach provided insights gathering information from sediments of different marine ecosystems. In the current work, co-occurrence fungal and bacterial interactions are presented gathering data from sediments of various sites, mainly in France. To take this effort further, the analysis of sediments from other different and numerous locations worldwide will allow to obtain a complete pattern of yet undescribed fungi and bacteria interactions as well as their diversity. Two strategies are proposed to make a robust analysis of co-occurrence network describing fungal and bacterial interactions: i) an in-situ sampling and analysis of different sediments from various locations, and ii) a metadata analysis taking advantage of already available information present in databases on the fungal and bacterial diversity in marine coastal sediments.

Several questions remain open about the transport of bacterial along the fungal mycelia in contaminated sediments. Whereas in this work we showed that selected isolated fungi effectively transport bacterial communities along hydrophobic patches in culture media. Thus, the uncultivable The results from this experiment will provide an insight of the non-cultivable diversity of bacteria able to be transported along hyphal translocation network without the selective pressure of the media. In addition to this, the microcosm will answer whether bacterial transport along fungal network can occur under water-filled environments.

Finally, all the data recollected from the project here presented, pave the way for implementing new strategies for the recovery of contaminated coastal marine environments, including: i) the development of bioreactors with fungi and bacteria selectively chosen for their capacity to up take and degrade PAHs; ii) the use of communication molecules (COVs) to stimulate fungi-bacteria interaction for enhancing PAHs degradation in situ; iii) the identification of key taxa of fungi and bacteria partners from contaminated marine ecosystems representing potential bioindicators to report the level of pollution and the recovery capacity.

Resumen

Los sedimentos marinos son medios ambientes heterogéneos con una variedad de características físico-químicas. Son el reservorio de una vasta diversidad microbiana que no ha sido totalmente explorada. Entre las comunidades microbianas, la interacción entre hongos y bacterias juega un papel clave en los procesos biogeoquímicos incluyendo los ciclos del sulfuro, nitrógeno y carbono. Sin embargo, las interacciones entre hongos y bacterias son constantemente alteradas por la presencia de hidrocarburos policíclicos aromáticos (HPAs), los cuáles son tóxicos, mutagénicos y carcinógenos. Los HPAs son moléculas resistentes y persistentes debido a su hidrofobicidad que les permite acumularse en los sedimentos donde la biodegradación es reducida debido a la reducción de su disponibilidad. En este contexto, el objetivo de esta tesis doctoral es profundizar en el entendimiento de las interacciones entre hongos y bacterias en los sedimentos costeros contaminados con HPAs. El análisis de redes de co-occurencia de los sedimentos costeros contaminados y no contaminados con HAPs, basados en las secuencias de los genes rRNA 16S y 18S (para bacterias y hongos, respectivamente), revelo que hay variantes de secuencias amplificadas (VSAs) provenientes de hongos que juegan un papel clave en la organización y ensamblaje de las comunidades microbianas estableciendo interacciones con VSAs de bacterias. La presencia de HPAs afecta dichas interacciones promoviendo un metabolismo anaeróbico. Las interacciones entre hongos y bacterias fueron también investigadas a través del cultivo de diversos hongos, de los cuales fueron aisladas y seleccionadas cepas fúngicas con capacidad de remover HAPs. De éstas cepas, Alternaria destruens F10.81 y Fusarium pseudoygamai F5.76 dieron evidencia de dos estrategias de captura y transporte intracelular de HAPs que involucran el almacenamiento citoplasmático o vacuolas, respectivamente. Dichos hongos fueron usados a su vez para evaluar la selección y el trasporte de comunidades bacterianas a través de una barrera de sedimento contaminada con HAPs. La diversidad de las comunidades bacterianas, basadas en el análisis de la secuencia de los genes 16S rRNA, revelo que existen bacterias seleccionadas de manera específica por la red de micelio fúngico. A su vez, cuatro biomarcadores específicos para Alternaria destruens F10.81 fueron identificados como Thalassospira sp., Spirochaeta litoralis, Arcobacter sp. y Vibrio sp. Sorprendentemente, Spirochaeta litoralis es una bacteria clasificada como estricta anaerobia sugiriendo la creación de zonas de anaerobiosis derivadas del desarrollo fúngico, lo cuál fue confirmado por la observación del consumo de oxígeno durante el crecimiento de las hifas de Alternaria destruens F10.81. Este resultado, fue consistente con la promoción del metabolismo anaeróbico encontrado en los análisis de redes de co-occurencia. Por lo tanto, nuevos conocimientos sobre el papel clave que juegan las interacciones entre hongos y bacterias en la organización de comunidades microbianas de sedimentos marinos costeros contaminados con HAPs son aportados, los cuales representan información útil para desarrollar estrategias de recuperación medioambiental.

Palabras clave: hongo, bacteria, diversidad microbiana, interacciones, red de transporte, hifósfera, HPA, contaminación, biodisponibilidad, sedimentos, aeróbico/anaeróbico.

Résumé

Les sédiments côtiers sont des environnements hétérogènes avec une variété de caractéristiques physico-chimiques. Ils sont le réservoir d'une vaste diversité microbienne, encore à explorer. Dans les communautés microbiennes, les interactions champignons-bactéries jouent un rôle clé dans plusieurs processus biogéochimiques, notamment le cycle du soufre, de l'azote et du carbone. Néanmoins, les interactions champignons-bactéries sont constamment affectées par l'apport de polluants, en particulier les hydrocarbures aromatiques polycycliques (HAP), qui sont toxiques, mutagènes et cancérigènes. Les HAP sont des molécules persistantes en raison de leur hydrophobicité, s'accumulant dans les sédiments où leur biodisponibilité pour la dégradation est réduite. Dans ce contexte, l'objectif de cette thèse est de mieux comprendre les interactions champignons-bactéries dans les sédiments côtiers contaminés par les HAP. L'analyse des réseaux de cooccurrence champignons-bactéries à partir de sédiments côtiers contaminés ou non par les HAP, basée sur les séquences des

Figure I. 1 .

 1 Figure I.1. Formation of marine sediments, sources and depositional pathways[START_REF] Maslin | ISOTOPES IN MARINE SEDIMENTS[END_REF].

Figure I. 2 .

 2 Figure I.2. Fungi and their ecological role in marine ecosystem[START_REF] Grossart | Aquatic fungi: targeting the forgotten in microbial ecology[END_REF].

  3 million tonnes of petroleum enters the marine environment each year (National Research Council (US) Committee on Oil in the Sea, 2003). Acute pollution incidents cause great public concern, notably ~600,000 tonnes of crude oil released after the Deepwater Horizon explosion in the Gulf of Mexico (Crone and Tolstoy, 2010) and approximately 5.74 million tonnes have been lost as a result of tanker incidents between 1970 and 2017 (Oil Tanker spill statistics, 2017). The impact of hydrocarbons, especially PAHs, on wildlife and fisheries may be longlasting (McGenity et al., 2012).

Figure I. 3 .

 3 Figure I.3. Structure of some 16 PAHs listed by the High Priority Pollutants by the Environmental Protection Agency (EPA)[START_REF] Ghosal | Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review[END_REF].

Figure I. 4 .

 4 Figure I.4. PAHs input, fate and deposit in marine ecosystems (adapted from Cardoso et al., 2016)

  dihydroxylation and cleavage leading to more intermediates and ultimate conversion to intermediates of the tricarboxylic acid cycle (van Der Meer et al., 1992; Kanaly and Harayama, 2000) (Figure I.5).

Figure I. 5 .

 5 Figure I.5. PAHs degradation pathway for bacteria or fungi[START_REF] Shahsavari | Phytoremediation and Necrophytoremediation of Petrogenic Hydrocarbon-Contaminated Soils[END_REF].

  PAHs bioavalability that in turn decreases its biodegradation. Nevertheless, several hydrocarbonoclastic halophilic and halotolerant microorganisms have been isolated from chronically hydrocarbon impacted sites close to petroleum industry facilities, most of them affiliated to the δproteobacteria class (Bruns and Berthe

  dispersal of bacteria(Simon et al., 2015) (Figure I.6). It has been demonstrated that mycelia-based bacterial dispersal stimulates contaminant biodegradation in situations where chemicals and/or bacteria are heterogeneously distributed(Worrich et al., 2016).

Figure I. 6 .

 6 Figure I.6. Fungal mycelia as vectors of motile bacteria dispersion and intracellular transport of PAHs via vacuoles(Furuno et al., 2012;[START_REF] Harms | Untapped potential: exploiting fungi in bioremediation of hazardous chemicals[END_REF].

(

  Harms and Wick, 2006) (Figure I.7). In oil-polluted sediments, fungi are likely primary degraders of high molecular weight hydrocarbons via secreted extracellular enzymes and work synergistically with oil-degrading bacteria(McGenity et al., 2012).

Figure I. 7 .

 7 Figure I.7. Fungi and bacteria advantages and interactions in PAHs degradation.

Figure

  Figure II.1). The module with the higher amount of ASVs was dominated by bacterial ASVs (M_1, Figure1) while the second module was mainly confined by fungal ASVs (M_3, Figure II.1). Such observations suggested that modules dominated by fungi have probably different ecological roles than modules dominated by bacteria. The other modules were smaller containing less than 10 nodes. These cooccurrences might reflect ASVs performing similarly or complementary functions, but they may also be caused by shared preferred environmental conditions[START_REF] Steele | Marine bacterial, archaeal and protistan association networks reveal ecological linkages[END_REF]. In agreement with previous works(Chaffron et al., 2010; Freilich et al., 2010; Faust and Raes, 2012), each module represents probably a specific ecological and/or functional niche.

Figure II. 1 :

 1 Figure II.1: Fungal-bacteria co-occurrence network in microbial community from coastal marine sediment. The co-occurrence network is based on Spearman positive correlations. Each node corresponds to fungal (red) or bacterial (blue) ASVs with a schematic icon corresponding to kingdom taxonomical classification. Edge lines represent significant co-occurrence relationships between nodes with a Spearman correlation of 0.7 and a p-value > 0.05. Modularity is represented with colours and characters (M_1 to M_15). The network parameters are indicated in the Table.

  occurrence networks were constructed using a set of 35 samples separated by no contaminated (17 samples) and PAHs contaminated (18 samples). The networks were constructed by data sets containing 2523 bacterial ASVs and 282 fungal ASVs for the no contaminated, and 2688 bacterial ASVs and 287 fungal ASVs for the PAHs contaminated. The networks were constructed with significant Spearman correlation coefficient > 0.75 and a p-value < 0.005 to select the strongest relations (Figure2). The resulting networks had a different number of bacterial nodes, being more abundant in nocontaminated sediments, even though both networks had a similar number of fungal nodes. The different amount of bacterial nodes modifies the ratio bacteria to fungi that correspond to 11.9 for no contaminated network and 4.3 for PAHs contaminated network. Such result suggested that the presence of PAHs reduce the fungi-bacteria interactions, affecting mainly the bacteria diversity probably by the selection of PAHs tolerant bacteria. In agreement with previous reports in soil, hydrocarbon contamination had an effect on the structure of the fungal-bacterial communities[START_REF] Bell | Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny[END_REF]. Both networks have similar average weight but they present shortest path significant differences (Figure II.2), showing less connections and more specific links for PAHs contaminated network. Thus, this observation suggested that there are specialist fungi-bacteria associations in PAHs contaminated sediments. The links observed in the PAHs-contaminated network probably play an important role in PAHs degradation, or at least these links insure a PAHs tolerance. Moreover, the strong links in this PAHs-contaminated network highlights the importance of fungal-bacterial interactions, suggesting that both microorganisms cooperate to overcome PAHs contamination.

Figure II. 2 .

 2 Figure II.2. Fungal-bacteria co-occurrence networks in microbial community from no contaminated and PAHs contaminated coastal marine sediments. The co-occurrence network is based on Spearman positive correlations. Each node represents fungal (red) or bacterial (blue) ASVs with a schematic icon corresponding to kingdom taxonomical classification. Edge lines represent significant co-occurrence relationships between nodes with a Spearman correlation of 0.75 and a p-value > 0.005. Edge size is proportional to the Spearman correlation coefficient. The network parameters are indicated in the Tables.

  were performed in Helmholtz Centre for Environmental Research -UFZ, Germany, with the collaboration of Lukas Y. Wick and his teamwork. The results obtained are presented in scientific publication format and were published in January 2021 in the Marine Pollution Bulletin under the title "Fungi in PAH-contaminated marine sediments: Cultivable diversity and tolerance capacity towards PAH".

1

  First, to explore the bacteria transport aptitudes required for the translocation via fungal hyphae, eight bacteria were isolated and challenged to use fungal networks to transport through gaps with different hydrophobicities (air, wet sand, PAHs contaminated sediment and wax) in media containing PAHs. Then, to evaluate bacterial selectivity by fungal mycelia from A. destrins F10.81 and F. pseudoygamai F5.76 used by bacteria as translocation networks through a PAHs contaminated gap, the diversity of bacterial communities was analyzed. The selectivity of bacteria by fungal translocation networks in sediments is for the first time described. The findings described are congruent with previous studies, which demonstrate bacterial selectivity along with fungal translocation networks[START_REF] Zhang | Signal beyond nutrient, fructose, exuded by an arbuscular mycorrhizal fungus triggers phytate mineralization by a phosphate solubilizing bacterium[END_REF]. To further characterize the selectivity of bacteria by fungal translocation networks, analysis that allow the targeting of specific biomarkers related to fungal and bacterial interactions were carried on. The results from the analysis showed the transport of aerobic and anaerobic bacteria, which was unexpected and suggest the creation of oxic and anoxic niches during the fungal development. To corroborate this hypothesis, analyses of oxygen depletation around the hypha were additionally performed. The information gather in this chapter, give an insight of fungal and bacterial interactions and transport under PAHs contamination stress. This work was done with the collaboration of Lukas Y. Wick, Bijing Xiong and their teamwork in Helmholtz Centre for Environmental Research -UFZ, Germany, as part of E2S and FunDeg project funded by PHC-PROCOPE exchanging program. The obtained results are presented in scientific article format that will be submitted in the ISME Journal (Interdisciplinary Journal of Microbial Ecology) for publication. Chapter IV: Marine fungal translocation network transport aerobic/anaerobic bacteria from PAHs contaminated sediments Université de Pau et des Pays de l'Adour, UPPA/E2S, IPREM UMR CNRS 5254, Bat. IBEAS, Pau, France 2 Helmholtz Centre for Environmental Research -UFZ, Department of Environmental Microbiology, Leipzig, 04318, Germany *Corresponding author: Robert Duran, Université de Pau et des Pays de l'Adour, E2S/UPPA, IPREM UMR CNRS 5254, BP 1155, 64013 Pau Cedex, France. E-mail: robert.duran@univ-pau.fr, tel: (+33)559407468. IV.1. Abstract This work present the first evaluation of bacterial transport along fungal mycelia through hydrophobic patches under conditions mimicking marine coastal sediments. The capacity of Alternaria destruens F10.81 and Fusarium pseudoygamai F5.76 strains to transport isolated bacteria through different gaps (air, water, sand, sediment) in PAHs containing media was first examined, revealing that PAHs tolerance and bacterial motility are required features for transport. The characterization of bacterial communities collected along the fungal mycelia showed that mycelia transport preferential bacteria indicating fungal selectivity relied by specific fungal and bacterial interactions occurring along the hyphosphere. Spirochaeta litoralis, known as strictly anaerobic bacteria was identified among the four specific bacterial strains transported by Alternaria destruens F10.81. Such result, suggests the formation of anoxic micro-niches during the hyphal development, which was confirmed by observing oxygen depletion around the hyphae during the fungal development. We thus advocate that aerobic bacteria are first transported at the oxic front of mycelia growth and then anaerobic bacteria are transported following the oxygen depletion by fungal growth. The results presented here show new insight of fungal-bacteria interactions in PAHs contaminated sediments paving the way for new questions related to unexplored inter-kingdom interaction mechanisms. Keywords: fungal translocation network, fungal-bacterial interactions, hyphal development, oxygen consumption, bacterial diversity, anaerobic bacteria

  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).

.3. 6 .

 6 DNA extraction and Illumina Miseq sequencing DNA from the setups at each sampling point and from samples of sediments from Etang de Berre and Canal Vieil were extracted with PowerSoil DNA Isolation Kit (MoBio Laboratories) according to the manufacturer's recommendations. PCR amplification of the V3-V4 hypervariable regions (344F-801R)

  Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76, with high and low removal PAHs capacity respectively (Álvarez-Barragán et al., 2021), were used as translocation networks to cross different physical barriers (Figure IV.1B). The bacterial transport along the translocation networks was observed only with motile bacteria able to tolerate the presence of pyrene (Table

Figure IV. 1 .

 1 Figure IV.1. Experimental design for bacteria (A, B) and bacterial communities (C, D) transport through translocation networks. A) Isolated bacteria translocated through physical barriers (air, wet sand, sediment, and wax) a. before inoculation, b. 48h after fungus inoculation, c. the bacteria inoculation, d. 10 days after bacterial inoculation, the presence of bacteria was observed after the gap. B) Pictures showing the different gaps used. C) Bacterial communities transport through fungal or glass fibbers translocation networks. The sampling points are indicated: sediment inoculum (1), before the gap (2), sediment gap (3), and after the gap (4). D) Pictures showing the experimental device after incubation in presence of fungi and glass fibbers.

  Table IV.1. Bacterial characteristics and their translocation capacities through the hyphae of F. pseudonygamai F5.76 (F) and A. destruens F10.58 (A).* Cultured along 16h at 25 °C in swimming and swarming media; ** Sea water minimal media added with 30 mg/L of pyrene as unique carbon source; *** sw-MM added with 10% LB and 30 mgL-1 of pyrene; F -F. pseudonygamai F5.76; A -A. destruens F10.58.

  on the translocation networks (Figure IV.2A). For Canal Vieil the only significant difference (PERMANOVA, p-value < 0.05) was observed between bacterial communities before and after the gap on the F. pseudonygamai F5.76 translocation network (Figure IV.2A). In contrast, for Etang de Berre bacterial communities were not different before and after the gap for fungal translocation networks (PERMANOVA, p-value > 0.05) while they were different for the glass fibber translocation network (PERMANOVA, p-value < 0.05) (Figure IV.2A). Consistently with alpha diversity analysis, it is likely that the selection of bacteria was dependent on the origin of microbial community and the translocation network. The composition of bacterial communities from Etang de Berre and Canal Vieil sediments were different (PERMANOVA, p-value < 0.001) (Figure IV.2B). Despite the fact that the bacterial communities of the inoculum from Etang de Berre and Canal Vieil were different irrespective of the translocation network, the bacterial communities were similar after the gap with the fungal translocation networks for both sediments (A. destruens F10.81, F. pseudonygamai F5.76, PERMANOVA, p-value > 0.05) (Figure IV.2B). Such observation further supports the selection of bacterial communities by the fungal translocation network, which was consistent with the study

Figure

  Figure IV.2. Comparison of bacterial communities by nonmetric multidimensional scaling (NMDS). The NMDSs, based on Bray-Curtis pairwise dissimilarity, were performed including all samples and then dispatched according to the translocation networks for representation. A) Comparison of bacterial communities from the different sampling points in each translocation networks with sediments from Canal Vieil and Etang de Berre. B) Comparison of bacterial communities obtained from Canal Vieil and Etang de Berre sediments in each translocation networks at inoculum and after the gap sampling points.

  were observed according to the translocation networks represented at low abundance after the gap (Figure IV.3), which might represent selection of specific genera related to FBI, consistent with alpha diversity observations.

Figure IV. 3 .

 3 Figure IV.3. Composition of the translocated bacterial communities obtained at the different sampling points with A. destruens F10.81, F. pseudonygamai F5.76, and glass fibbers translocation networks from sediments of A) Canal Vieil and B) Etang de Berre. The analysis was performed at the genus level applying a threshold similarity of 97% for OTU identification. Only genera with more than 0.1% of relative abundance are presented.

  When comparing fungal translocation networks with glass fibbers, eight different biomarkers were identified for glass fibbers and five for A. destruens F10.81 (Figure IV.4 A, B). When comparing A. destruens F10.81 and F. pseudonygamai F5.76 translocation networks, one biomarker was found for F. pseudonygamai F5.76 (ASV affiliated to Pseudomonas sp.) and six biomarkers for A. destruens F10.81 (Figure IV.4C). The different number of specific biomarkers for fungal translocation networks can be explained by their different pyrene removal capacities (Álvarez-Barragán et al., 2021), even if other parameters cannot be excluded. Interestingly four ASVs identified as biomarkers for A. destruens F10.81 were found common when comparing either with glass fibbers (Figure IV.4B) or F. pseudonygamai F5.76 (Figure IV.4 C), suggesting that they have been specifically selected along the A. destruens F10.81 translocation network. These ASVs are related to Thalassospira sp., Spirochaeta litoralis, Arcobacter sp. and Vibrio sp. All these genus have been detected in marine sediments and with

Figure IV. 4 .

 4 Figure IV.4. Comparison of translocated bacterial communities according to the translocation network.The linear discriminant analysis effect size (LEfSe) identified significant differential abundance distribution of bacterial ASVs, from bacterial communities obtained after the gap, between A. destruens F10.81. Hyphae and glass fibbers (A), F. pseudonygamai F5.76 hyphae and glass fibbers (B), and between both fungi (C). The analysis was performed considering bacterial communities obtained after the gap with both Etang de Berre and Canal Vieil sediments. The stars indicates ASVs biomarkers identified for A. destruens F10.81(orange) and glass fibbers (purple) present in the two comparisons performed for each translocation network.

Figure IV. 5 .

 5 Figure IV.5. Oxygen depletion during fungal growth. A) Schematic view of the microcosm used for mycopshere oxygen mapping and B) spatially distinct oxygen gradient (0%-100%) was observed in the mycopshere of A. destruens F10.81. Anoxic environment (indicated by blue colour) was observed in the mycosphere where dense hyphal networks were established.

  Such result suggested that the oxygen depletion during the hyphal development create anoxic niches allowing the translocation of facultative and anaerobic bacteria. This capacity is of environmental relevance in PAH contaminated coastal sediments allowing anaerobic and aerobic bacteria to be transported through different hydrophobic patches via fungal network. We advocate that aerobic bacteria are transported in the rich oxygen apical hyphae zone while anaerobic bacteria follow after oxygen depletion by fungal growth(Figure IV.6).

Figure

  Figure IV.6. Fungal translocation of aerobic and anaerobic bacteria. Schematic representation of the hyphosphere showing oxic (red) and anoxic (blue) zones formed during the hyphal growth (t1 and t2) across the PAHs contaminated sediment gap in which aerobic (green) and anaerobic (purple) translocate.

  fungal communities and their associated bacterial communities were not assessed. In an experimental microbial ecology approach, microcosm experiments might help to mimic as closer as possible marine coastal sediment ecosystem under laboratory conditions in order to further study fungi-bacteria interactions (Figure V.1).

Figure V. 1 .

 1 Figure V.1. Schematic representation of the experimental setup designed to analyse bacterial communities transported by fungal translocation network on sediments polluted with PAHs. a) Frontal view: The microcosm consisting a petri dish of 5 cm diameter with a fill-up with seawater media agar (to keep osmolality and offer physical support) except for an empty compartment of 3x1 cm in the centre. b) Lateral view: The central compartment is divided with paper membranes that can keep separated sediments with different characteristics. All the sediments come from the same source but received different treatments: a) nothing, b) autoclaved and PAHs input, c) autoclaved. In addition, the complete system is covered with seawater to emulate a water-filled environment.

  ARNr 16S et 18S (respectivement pour les bactéries et les champignons), a révélé que des variants spécifiques de séquences amplifiées (ASV) fongiques jouent un rôle clé dans l'organisation d'assemblages de communautés microbiennes en établissant des interactions avec des ASV bactériens. La présence d'HAP affecte ces interactions favorisant le métabolisme anaérobie. Les interactions champignons-bactéries ont été étudiées afin d'explorer la diversité fongique cultivable qui a permis d'isoler, d'identifier et de sélectionner des souches fongiques des environnements marins pour leur capacité à éliminer les HAP. Alternaria destruens F10.81 et Fusarium pseudoygamai F5.76 ont montré des stratégies différentes pour l'absorption des HAP qui impliquent respectivement le transport à travers le cytoplasme ou via des vacuoles. Ces souches fongiques ont été utilisées pour évaluer la sélection et le transport des communautés bactériennes à travers des zones contaminées par les HAP. Des analyses de diversité des communautés bactériennes, basées sur des séquences des gènes 16S ARNr, ont révélé que des bactéries spécifiques étaient sélectionnées par le réseau mycélien fongique. Par ailleurs, quatre biomarqueurs spécifiques à Alternaria destruens F10.81 ont été identifiés : Thalassospira sp., Spirochaeta litoralis, Arcobacter sp. et Vibrio sp. De manière surprenante, Spirochaeta litoralis est connue comme une bactérie anaérobie suggérant que le développement de mycélium fongique produit des zones anaérobies, confirmée par l'observation de la consommation d'oxygène pendant le développement des hyphes d'A. destruens F10.81. Un tel résultat était cohérent avec la promotion du métabolisme anaérobie décrit avec le réseau de cooccurrence champignons-bactéries. Ainsi, de nouvelles informations sur le rôle clé des interactions fongiques et bactériennes sur l'organisation des communautés microbiennes dans les sédiments côtiers contaminés par les HAP sont fournies, et représentent des informations utiles pour développer des stratégies de bioremédiation environnementale. Mots clés: champignons, bactéries, diversité microbienne, interactions, réseau de translocation, hyphosphère, HAP, contamination, biodisponibilité, sédiments, aérobie/anaérobie.

Table I .1. Identified fungi in marine environments.

 I

	Phylum	Genera and specie	Analysis	Source and site	Reference
	Ascomycota	Aspergillus niger, Aspergillus terrus,	Isolation and	Coastline from	Elshafie et
		Aspergillus nidulans, Cladosporium	culture	Gulf of Oman	al., 2007
		cladosporoides, Penicillium,
		Pacelomyces, Aspergillus ochraceus,
		Alternaria alternata, Penicillium
		chrysogenum
	Ascomycota	Acremonium, Aspergillus, Cordyceps,	Culture and	Deep subseafloor	Rédou et
	Basidiomycota	Eurotium, Exophilia, Cladophialophora,	enrichment	from Cantebury	al., 2015
		Cladosporium, Fusarium, Microascus,		Basin, New
		Oidiodendron, Paecilomyces,		Zealand
		Penicillium, Phialophora,
		Purpureocillium, Sarocladium,
		Bjerkandera, Sistotrema, Trametes
	Ascomycota	Trichoderma harzianum, Trichoderma	Isolation and	Marine	Ahumada-
	Basidiomycota	deliquescens, Penicillium crustosum,	culture	sediments from	Rudolph et
		Talaromyces atroroseus, Rhodotorula		Aysén	al., 2019
		mucilaginosa		Providence, Chile
	Ascomycota	Acremonium, Aspergillus, Eurotium	Isolation and	Marine	Mouton et
	Basidiomycota	amstelodami, Paraconiothyrium	culture	sediments from	al., 2012
		cyclothyrioides, Penicillium,		Sta. Helena Bay,
		Trichoderma, Yarrowia lipolytica,		South Africa
		Rhodotorula mucilaginosa
	Ascomycota	Neurospora, Emerichella,	Isolation and	Coastal marine	Babu et al.,
	Zygomycota	Sacharamyces, Aspergillus, Penicillium,	culture	water and	2010
		Verticillium, Alternaria,		sediments from
		Cephalosporium, Cladosporium,		South-East Coast,
		Curvularia, Drechslera, Nigrospora,		India
		Fusarium, Ascochyta, Mucor, Rhizopus
	Basidiomycota	Ustilaginomycetes, Basidiomycetes	DNA analysis from	Deep sea	Edgcomb
			18S rRNA gene	sediments from	et al., 2011
				Peru
	Ascomycota	Cryptococcus, Candida, Trichosporon,	Clones identified	Deep sea	Zhang et
	Basidiomycota	Rhodotorula, Aspergillus, Alternaria,	by ITS gene	sediments from	al., 2014
	Zygomycota	Cladophialophora, Cladosporium,		East Indian
		Fusarium, Geomyces, Trichoderma,		Ocean
		Galactomyces, Sterigmatomyces,
		Basidiomycete, Dipodascus,
		Guehomyces, Horteae,
		Sporobolomyces, Eurotium, Hypocrea,
		Leptospheria, Mortierella, Phoma,
		Xeromyces, Rhizoscyphus
	Ascomycota	Penicillium	Isolation and	Sediments	Amer et
			culture	ranging between	al., 2019
				0.5 to 1m from
				Alexandria, Egypt
	Ascomycota (62 OTUs)	DNA extraction	Marine	Zhang et
	Basidiomycota (26 OTUs)	and 454	sediments from	al., 2015
	Glomeromycota (1 OTU)	pyrosequencing	High Artic
	Unkwnown (21 OTUs)	ITS1F-ITS4	Kongsfjorden,
	Zygomycota (1 OTU)		Svalbard
	Chrytridiomycota (2 OTUs)

Table I .2. Fungi detected in contaminated marine environments Phylum Genera and specie Analysis Source and site Contamination Reference

 I

	Ascomycota	Fusarium, Scopulariopsis,	Isolation	Beaches from Gulf	Crude oil	Simister et
		Aspergillus	and	of Mexico, Mexico		al., 2015
			culture
	Ascomycota	Aspergillus, Cladosporium,	Isolation	Sediments	Pyrene	Ravelet et
	Zygomycota	Coniothyrium, Curvularia,	and			al., 2000
		Fusarium, Getrichum,	culture
		Gliocladium, Monocillum,
		Penicillium, Phialophora,
		Scopulariopsis, Trichoderma,
		Absidia, Gongronella,
		Mucor, Rhizomucor
	Basidiomycota Tinctoporellus,	Isolation	Marine derived	Pyrene	Vieira et al.,
		Marasmiellus, Peniophora	and	from Brazilian	Benzo(α)pyrene	2018
			culture	sponges
	Ascomycota	-	Illumina	Seawater from Foz	Benzene,	Campeão et
	Microsporidia		sequencing	do Amazonas	Toluene,	al., 2017
				Barreirinhas, Brazil	Ethylbezenes,
					Xylene, PAHs,
					Alkanes
	Ascomycota	Aspergillus, Candida,	Isolation	Water and	Crude oil	Chikere and
	Zygomycota	Penicillium, Rhizopus,	and	sediments from		Azubuike,
		Saccharomyces,	culture	Rivers State,		2014
		Cladosporium*, Fusarium*,		Nigeria
		Mucor*
		*just in sediments
	Ascomycota	Cladosporium	Isolation	Marine derived	Anthracene	Birolli et al.,
			and	from Sao Paulo	Acenaphthene	2018
			culture	State Coast, Brazil	Phenanthrene
					Pyrene
					Anthraquinone
					Fluorene
					Fluoranthene
	Ascomycota	Aspergillus, Penicillium	Isolation	Mangrove	Crude oil	Ghizelini et
			and	sediments from		al., 2019
			culture	Sepetiba Bay and
				Guanabara Bay,
				Brazil

Fungal-bacterial network in PAHs contaminated coastal marine sediment Joyce Álvarez-Barragán, Cristiana Cravo-Laureau, Robert Duran* Université

	Chapter II. Fungal-bacterial co-occurrence in PAHs contaminated coastal
	marine sediment: network analysis
	II.2.
	Science and Pollution Research

de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM , Pau, France *Corresponding author: Robert Duran, Université de Pau et des Pays de l'Adour, E2S/UPPA, IPREM UMR CNRS 5254, BP 1155, 64013 Pau Cedex, France. E-mail: robert.duran@univ-pau.fr, tel: (+33)559407468.

Table II .1. Fungal-bacterial interactions from network analysis from PAHs contaminated sediments with a Spearman's correlation above 0.82 and p-value < 0.005.

 II occur in PAHs contaminated coastal sediments interacting with diverse bacterial populations creating assemblages allowing to both, fungi and bacteria, to survive in hazardous conditions. PAHs contamination alters the fungi-bacteria interactions, resulting in the selection of the stronger interactions. Interestingly, the fungal Chytridiomycota phylum was found to interact with anaerobic bacteria, which suggest that anaerobic metabolism of PAHs has a central role in the microbial community structure. Further studies are required to fully understand these interactions and their functional role in response to PAHs contamination.[START_REF] Zhang | Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden[END_REF]. Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic). Sci. Rep. 5.Zhang, Y., Kastman, E.K., Guasto, J.S., and Wolfe, B.E. (2018). Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes. Nat. Commun. 9.

	II.2.5. Conclusion
		Fungal taxonomy	Fungal key	Bacterial key	Bacterial taxonomy
	Epsilonbacteraeota, Campylobacteria, Campylobacterales, Thiovulaceae, Sulfurimonas Zhao, B., Wang, H., Li, R., and Mao, X. (2010). Thalassospira xianhensis sp. nov., a polycyclic aromatic B3333 B9688 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae, Actibacter Desulfosarcina B8551 Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobacteraceae, hydrocarbon-degrading marine bacterium. Int. J. Syst. Evol. Microbiol. 60, 1125-1129.
		Unclassified	F966	B4096 Proteobacteria, Gammaproteobacteria, Gammaproteobacteria Incertae Sedis B3979 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae
				B9017 Bacteroidetes, Bacteroidia, Chitinophagales, Saprospiraceae
				B4008	Acidobacteria, Thermoanaerobaculia, Thermoanaerobaculales, Thermoanaerobaculaceae, Subgroup 10
				B4403 Planctomycetes, Phycisphaerae, MSBL9, SG8-4
				B9329 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae, Lutibacter
				B9290	Proteobacteria, Alphaproteobacteria, Rhizobiales, Rhizobiales Incertae Sedis, Anderseniella
	No contaminated	Chytridiomycota, Incertae Sedis, Chytridiomycetes, Lobulomycetales, Lobulomycetaceae	F876	B2840 Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobacteraceae B5700 Cyanobacteria, Oxyphotobacteria, Chloroplast, Cylindrotheca closterium B8202 Bacteroidetes, Bacteroidia, Cytophagales, Cyclobacteriaceae B6093 Cyanobacteria, Oxyphotobacteria, Chloroplast B3633 Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobacteraceae, Desulfococcus B1112 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae, Eudoraea
				B1060 Proteobacteria, Gammaproteobacteria
				B1415 Cyanobacteria, Oxyphotobacteria, Chloroplast
		Dikarya,		B5672 Fusobacteria, Fusobacteriia, Fusobacteriales, Fusobacteriaceae, Psychrilyobacter
		Ascomycota,
		Pezizomycotina,		B3401 Proteobacteria, Alphaproteobacteria, Rhodobacterales, Rhodobacteraceae
		Eurotiomycetes, Eurotiales,	F903	B4227 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae, Lutimonas
		Aspergillaceae, Penicillium		B9235 Bacteroidetes, Bacteroidia, Flavobacteriales, Flavobacteriaceae, Winogradskyella
		Chytridiomycota,
		Incertae Sedis,
		Chytridiomycetes,	F373	B566	Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobulbaceae
		Lobulomycetales,
		Lobulomycetaceae
		Chytridiomycota,		B5835 Fusobacteria, Fusobacteriia, Fusobacteriales, Fusobacteriaceae, Propionigenium
		Incertae Sedis,		B3980 Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobulbaceae
	PAHs contaminated	Chytridiomycetes, Lobulomycetales, Lobulomycetaceae Dikarya, Ascomycota, Sordariomycetes, Hypocreales Pezizomycotina,	F373 F298	B2278 Bacteroidetes, Bacteroidia, Bacteroidales, Prolixibacteraceae B3980 Proteobacteria, Deltaproteobacteria, Desulfobacterales, Desulfobulbaceae
		Unclassified	F380	B1729 Proteobacteria, Alphaproteobacteria, Rhodobacterales, Rhodobacteraceae

Fungi

II.2.6. Acknowledgment

We would like to thank all partners of the MELODY group for their useful discussions. We acknowledge the financial support from CNRS programme LEFE-EC2CO through TOTEM project, and the French National Research Agency (ANR) through DHYVA (06-SEST-009) and DECAPAGE (CESA 006 01) projects. JAB received a fellowship from México CONACyT number 326250/471689.

  5 µL AmpliTaq Gold 360 Master Mix 2X (Thermo Fisher Scientific, USA). The

	amplification was performed through 35 cycles of 95 °C (45 s), 58 °C (45 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). Amplified
	fragments were sequenced at the Eurofins platform (France). Sequence data were edited using
	Chromas Pro version 1.34. For identification, 16S rRNA sequences were compared with NCBI (National
	Centre for Biotechnology Information; http://www.ncbi.nlm.nih.gov) database. The sequences
	determined in this study have been submitted to the NCBI database and assigned Accession
	numbers: OK128316 to OK128323.

Table IV .2. Alpha diversity indices of the translocated bacterial communities obtained at the different sampling points transported along different translocation networks

 IV

										Canal Vieil
	Indices	In situ	Cultivable	1	Inoculum	2	A. destruens F10.58 Before 2 Gap 2	After	2	Inoculum	2	F. pseudonygamai F5.76 Before 2 Gap 2	After	2	Inoculum	2	Glass fiber Before 2 Gap	2	After	2
	Reads	32 071 ± 1 839 37 147 ± 3 504	27 007 ± 2 796 41 234 ± 3 659 35 335 ± 1 367 37 752 ± 7 157 16 819 ± 12 089 32 294 ± 3 416 33 735 ± 4 715 38 393 ± 5 366 35 395 ± 5 536 35 264 ± 7 549 32 790 ± 1 520 38 183 ± 3 282
	Trimmed sequences	11 107 ± 1 186 16 415 ± 1 442	13 563 ± 2 287 24 005 ± 2 965 21 142 ± 941 23 301 ± 3 487 6 295 ± 5 035		16 605 ± 3 743 18 554 ± 3 233 27 196 ± 2 457 17 680 ± 4 013 22 502 ± 4 570 18 459 ± 858 23 127 ± 3 501

4.94 ± 0.16 2.66 ± 0.27 3.14 ± 0.07 0.7 ± 0.21 0.18 ± 0.09 0.23 ± 0.17 3.42 ± 0.05 1.36 ± 0.92 1.31 ± 0.2 0.43 ± 0.48 2.43 ± 0.32 1.32 ± 0.5 1.05 ± 0.83 0.46 ± 0.24

IV.6. Acknowledgments

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 México CONACyT number 326250/471689 and a mobility grant from the centre of excellence E2S at Pau University. EXPLORING FUNGI-BACTERIA INTERACTION IN PAH CONTAMINATED COASTAL SEDIMENT Joyce ÁLVAREZ BARRAGÁN -23/11/2021

Abstract

Coastal sediments are heterogeneous environments with a variety of physical-chemical characteristics. They are the reservoir of a vast microbial diversity, yet to be explored. In the microbial communities, the fungi-bacteria interactions play a key role in several biogeochemical processes including sulphur, nitrogen and carbon cycling. Nevertheless, the fungibacteria interactions are constantly affected by the input of pollutants, particularly polycyclic aromatic (PAHs) compounds, which are toxic, mutagenic and carcinogenic. PAHs are persistent molecules due to their hydrophobicity accumulating in sediments where their bioavailability for degradation is reduced. In this context, the aim of this PhD thesis is to better understand the fungi-bacteria interactions in coastal sediments contaminated with PAHs. The analysis of fungi-bacteria cooccurrence networks from coastal sediments contaminated or not with PAHs, based on 16S and 18S rRNA barcoding sequences (for bacteria and fungi respectively), revealed that specific fungal amplified sequence variants (ASVs) play a key role in the organization of microbial community assemblages by establishing interactions with bacterial ASVs. The presence of PAHs is affecting these interactions promoting anaerobic metabolism. The fungi-bacteria interactions were further investigated by exploring the fungal cultivable diversity that allowed to isolate, identify, and select fungal strains from marine environments for their capacity to remove PAHs. Alternaria destruens F10.81 and Fusarium pseudoygamai F5.76 showed different strategies for PAHs uptake that involve either the transport through the cytoplasm or via vacuoles, respectively. These fungal strains were used to evaluate the selection and transport of bacterial communities through PAHs contaminated gap. Diversity analyses of bacterial communities, based on 16S barcoding sequences revealed that specific bacteria were selected by the fungal mycelia network. Moreover, four biomarkers specific to Alternaria destruens F10.81 were identified as Thalassospira sp., Spirochaeta litoralis, Arcobacter sp. and Vibrio sp. Surprisingly, Spirochaeta litoralis is known as anaerobic bacteria suggesting that fungal mycelia development produce anaerobic zones, further confirmed by the observation of oxygen consumption during the hyphal development of A. destruens F10.81. Such result was consistent with the promotion of anaerobic metabolism described with the fungi-bacteria co-occurrence network. Thus, new insights on the key role of fungal and bacterial interactions have on the organization of microbial communities in coastal sediments contaminated by PAHs are provided, which represent useful information to develop strategies for environmental recovery.