A Rogani

P Navarro

S Marguet

J-F Ferrero

Study of post-impact behaviour of thin carbon/epoxy laminates subject to fatigue tensile loading

Keywords: Woven composites, fatigue, digital image correlation, explicit F.E modelling, semi-continuous approach

This article concerns the experimental and numerical study of post-impact damage propagation in thin carbon/epoxy woven composite laminates loaded in fatigue tension. Low velocity normal drop weight impact tests are first performed. Post-impact fatigue tensile tests are then carried out. They are controlled in displacement. The monitoring is based on Digital Image Correlation and RX tomography. The influence of the impact energy and the tensile fatigue loading on the post-impact damage propagation is studied. The damage propagation is governed by matrix damage, with the emergence of tows/resin splittings and intra-tows crackings as well as delamination when the plies have different orientations. When the impact energy or the displacement level increases, the post-impact damage initiates sooner and propagate faster. In some cases, that can lead to a quasi-instantaneous failure identical to that observed for quasi-static tensile loading. The FEM based semi-continuous approach, initialy developped for the modelling of impact damage, is extended to fatigue loading for carbon/epoxy woven laminates.

Fatigue damage laws, based on experimental observations are implemented.

The modelling well correlates the experimental results in terms of damage

Introduction

This article deals with the study of the post-impact behaviour of thin carbon/epoxy woven composite laminates under fatigue tensile loading. This paper is dedicated to the experimental and numerical study of samples submitted to a fatigue tensile loading after having been impacted with a low velocity in a drop tower device.

Carbon woven composite structures are widely used, especially in the field of aeronautics. However, these structures are known to be very sensitive to impact loadings. In fact, an impact can generate matrix cracking, delamination and fibre breakage. The impact can perforate the structure or the damages can be barely detectable. In any case, that induces a decrease of the mechanical properties and a loss of the strength. More, the damages can propagate if the structure is loaded after having been impacted, what can lead to the final failure.

For all these reasons, understanding and identifying the post-impact damage propagation phenomenons that occur in a carbon woven composite structures loaded with a fatigue tensile loading is important.

In general, fatigue studies on composite structures are conducted at a macroscopic scale in order to obtain the residual mechanical properties of the laminate such as the residual strength [START_REF] Cantwell | Post-impact fatigue performance of carbon fibre laminates with non-woven and mixed-woven layers[END_REF][START_REF] Ding | Effect of impact and fatigue loads on the strength of plain weave carbon-epoxy composites[END_REF][START_REF] Kang | Fatigue life prediction of impacted carbon/epoxy laminates under constant amplitude loading[END_REF][START_REF] Vallons | Impact and post-impact properties of a carbon fibre non-crimp fabric and a twill weave composite[END_REF][START_REF] Komus | Fatigue behaviour of a barely visible impact damaged carbon fibre reinforced epoxy laminate[END_REF], the residual strain or the residual stiffness [START_REF] Sudevan | Post-impact fatigue response of cfrp laminates under constant amplitude and programmed falstaff spectrum loading[END_REF]. Other authors try to characterize the fatigue behaviour of the material through the establishment of a Wöhler curve [START_REF] Komus | Fatigue behaviour of a barely visible impact damaged carbon fibre reinforced epoxy laminate[END_REF].

The visualization of the damage propagation is sometimes performed at a microscopic scale.

Existing studies concerns mainly compression-compression loading [START_REF] Beheshty | An empirical fatigue-life model for high-performance fibre composites with and without impact damage[END_REF] or tension-compression loading [START_REF] Melin | Fatigue testing and buckling characteristics of impacted composite specimens[END_REF][START_REF] Kimpara | Post-Impact Fatigue Behavior of Woven and Knitted Fabric CFRP Laminates for Marine Use[END_REF][START_REF] Garnier | Fatigue behavior of impacted composite structures[END_REF] rather than tension-tension loading.

Indeed, the delamination that can be generated by impact can provoke local buckling when loaded in compression that could lead to an unstable propagation of the damage [START_REF] Davies | Polymer Composites in the Aerospace Industry[END_REF]. For instance, Melin et al. [START_REF] Melin | Fatigue testing and buckling characteristics of impacted composite specimens[END_REF] concluded that, during tension-compression fatigue loading on impacted carbon/epoxy samples, the compressive part of the cycle had a more important role in failure than the tensile part. More, Beheshty et al. [START_REF] Beheshty | An empirical fatigue-life model for high-performance fibre composites with and without impact damage[END_REF] noticed that the effects of impact damage on carbon/epoxy laminates were more severe for compressioncompression fatigue loading than for tension-compression loading.

However, few studies concern the influence of an impact on the fatigue performances of a carbon woven composite under tensile loading. Cantwell et al. [START_REF] Cantwell | Post-impact fatigue performance of carbon fibre laminates with non-woven and mixed-woven layers[END_REF] have shown that substituting UD carbon plies by woven plies in a laminate could improve the fatigue behavior of the composite structure and Ding et al. [START_REF] Ding | Effect of impact and fatigue loads on the strength of plain weave carbon-epoxy composites[END_REF] have shown that the increase of the impact energy lead to a decrease of the residual strength of the laminate. More, the residual stiffness decreases with the impact energy [START_REF] Sudevan | Post-impact fatigue response of cfrp laminates under constant amplitude and programmed falstaff spectrum loading[END_REF]. It decreases in three steps : an initial fast decrease, a stable state which indicates the beginning of the transverse cracking and a final fall just before the failure.

Concerning the monitoring of the fatigue tests, various techniques are used in the literature to follow the damage during the fatigue test. For example, digital image correlation (DIC) [START_REF] Ambu | Investigation of the effect of damage on the strength of notched composite laminates by digital image correlation[END_REF][START_REF] Leser | Fatigue crack closure analysis using digital image correlation[END_REF][START_REF] Iadicola | 2d dic crack tip tracing under mode i loading[END_REF][START_REF] Conway | Multiscale behavior of fused deposition additively manufactured thermoplastic cellular materials[END_REF], acoustic emission [START_REF] Rosa | Post-impact static and cyclic flexural characterisation of hemp fibre reinforced laminates[END_REF] or self-heating methodology [START_REF] Gornet | A study of various indicators to determine the fatigue limit for woven carbon/epoxy composites under self heating methodology[END_REF]. Ambu et al. [START_REF] Ambu | Investigation of the effect of damage on the strength of notched composite laminates by digital image correlation[END_REF] monitored their tension-tension fatigue tests on CFRP samples with DIC. They found that DIC was a useful to identify the roles and the influence of each failure modes on the residual properties of notched samples, the mechanisms of damage development and the strain distribution around a hole. More, De Rosa et al. [START_REF] Rosa | Post-impact static and cyclic flexural characterisation of hemp fibre reinforced laminates[END_REF] made a correlation between the amplitude of acoustic emission and the type of damage occurring in the laminate. Gornet et al. [START_REF] Gornet | A study of various indicators to determine the fatigue limit for woven carbon/epoxy composites under self heating methodology[END_REF] made also a link between heating effects and damage mechanisms during fatigue loading.

Concerning the modelling of post-impact damage propagation in fatigue, the literature on the subject is barely existing. Most of the studies have been conducted on laminates with an initial defect such as notches, holes or delamination. Continuum damage modelling (CDM) is often used to model resin damaging [START_REF] Miot | Rupture de structures composites stratifiées sous chargements statique et de fatigue[END_REF][START_REF] Hochard | A generalized damage model for woven ply laminates under static and fatigue loading conditions[END_REF][START_REF] Maire | A new formulation of continuum damage mechanics (cdm) for composite materials[END_REF][START_REF] Marcin | Modélisation du comportement, de l'endommagement et de la rupture de matériaux composites à renforts tissés pour le dimensionnement robuste de structures[END_REF]. The principle is to degrade the stiffness matrix by one or several damage variables, calculated through thermodynamical forces. Some authors use empirical criterions, introduced by Whitney and Nuismer [START_REF] Whitney | Stress fracture criteria for laminated composites containing stress concentrations[END_REF], such as the point stress criterion (PSC) and the average stress criterion (ASC). Miot [START_REF] Miot | Rupture de structures composites stratifiées sous chargements statique et de fatigue[END_REF] used the ASC method for holed and notched carbon laminates. The mean stress is calculated into a characteristic volume in order to evaluate the damage propagation. But, even if the implementation of these criterions is simple, they are very dependant on the testings parameters.

Concerning the delamination modelling, cohesive elements, introduced by Hilleborg at al. [START_REF] Hillerborg | Analysis of crack formation and crack growth in concrete by means of fracture mechanics and finite elements[END_REF], are widely used for impact loading. Robinson et al. [START_REF] Robinson | Numerical simulation of fatigue-driven delamination using interface elements[END_REF] used it for the modelling of delamination propagation in fatigue.

These elements are also used in some studies to model the matrix crackings propagation. For example, Hallett et al. [START_REF] Hallett | An experimental and numerical investigation into the damage mechanisms in notched composites[END_REF] used them for a tensile simulation on a holed sample. For this purpose, they have first noted the position of the crackings after an experimental test. Then, they meshed the sample placing the cohesive elements on potential paths of the crack propagation.

This paper focuses on the post-impact damage propagation in three thin carbon/epoxy woven composite laminates. In a first time, the objective is to investigate the propagation phenomenons specific to carbon/epoxy thin woven laminates. Low velocity impacts are first performed with a drop weight device. Then fatigue tensile tests are carried out on the impacted samples.

Impact energies and levels of loading are varied. An analysis of the damage propagation scenario is based on measures from Digital Image Correlation and RX tomography. In a second time, the semi-continuous approach, already validated for post-impact quasi-static tensile loading on impacted carbon/epoxy thin woven laminates [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part ii: Numerical study[END_REF], is extended to fatigue loading. The innovative idea is to use the same model to calculate the damage due to an impact and its propagation. To do so, developments, based on the experimental observations are implemented. A strategy is developed in order to perform fatigue simulation on impacted samples in the explicit finite element code Radioss. Finally, the results of the simulation are compared to the experimental ones.

Experimental study

In this section, the experimental work is described. As it is performed in several steps (manufacturing, impact tests, fatigue tests, analyse) and for the seek of clarity, the experimental path is represented on the Figure 1.

Material and samples

In this study, three thin carbon/epoxy woven laminates configurations are investigated. The material is the G963/913 5-harness satin prepreg provided by Hexcel. These three configurations, presented (Table 1), are commonly used in some parts of aeronautical structures, for instance helicopter blades.

Configuration C0C0 is made up of two carbon plies oriented at (0/90) • , configuration C45C45 is made up of two carbon plies oriented at ±45 • and configuration C45C0 is made up of one ply oriented at (0/90) • and one ply oriented at ±45 • .

The samples have been manufactured using a hot platen press with a curing temperature of 135°C and a pressure of 7 bars. The curing time was set to 90 minutes. The presence of defects has been checked with RXtomography and no voids have been observed.

The sample geometry has been set in order to perform low velocity impact test and fatigue tensile tests on the same specimen. It is presented on the Figure 2. The dimensions are 100 mm x 300 mm with a thickness of 0.71 mm. The tabs are made with three plies of glass/epoxy woven oriented at ±45 • .

Low velocity impact tests

The impact tests have been described in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF]. They have been conducted with the drop weight device presented on the Figure 3. The impactor has a 16mm diameter steel head, with a mass of 2 kg. Multiple impact due to the rebound is prevented. The samples are clamped on a 75mmx125mm rectangular frame. Impact velocities and corresponding impact energies are varied. Five impact energies have been tested (Table 2). They have been chosen to have several levels of damage for each configuration in order to study how the propagation initiates. These impact tests have been carried out in the same experimental campaign than presented in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF]. The damage shapes and sizes are very repeatable. The fracture surfaces of the impacted samples are presented on the For the C0C0 and C45C45 configurations, cross-shaped fibres breakages oriented in the bundles directions are observed in the upper and lower plies.

For the C45C0 configuration, the fibres failure are oriented at (0/90) • in the lower ply and are oriented with an angle that varies between ±45 • and (0/90) • in the upper ply. Indeed, the damage propagation in the upper ply is influenced by the directions of the lower ply, as it has been shown in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF] that the damage initiates in this lower ply. The size of the damages increase with the energy of impact until an energy of 4 J for which the impactor totally perforates the sample. The load ratio is defined as R = ∆L dyn /∆L sta = 0.9. The level of the fatigue load is set as a function of the break displacement in quasi-static tension ∆L rupt QS measured in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF] : ∆L sta = 25% ∆L rupt QS for C45C45 and ∆L sta = 40% ∆L rupt QS for C0C0 and C45C0.

The reaction load is measured during the test. The strain fields of the samples are measured by the use of Digital Image Correlation (DIC). To do so, the tests are recorded with two cameras, filming the front and the back face of the sample (Figure 7).

The influence of the impact energy and fatigue cycle level on the postimpact damage propagation is studied. The values are gathered in the Table 3.

Three tests have been conducted for each configuration in order to check the repeatability.

Monitoring of the damage propagation

During the fatigue tests, the damage propagation is monitored with DIC in order to study the progression depending on the number of cycles.

To do so, the correlation quality factor Sigma calculated by the DIC is used. The more its value decreases, the more the correlation quality is high.

When damage appears in the sample, the value of the quality factor increases [START_REF] Leser | Fatigue crack closure analysis using digital image correlation[END_REF][START_REF] Iadicola | 2d dic crack tip tracing under mode i loading[END_REF][START_REF] Conway | Multiscale behavior of fused deposition additively manufactured thermoplastic cellular materials[END_REF]. For instance, Figure 9 shows the damage of a an impacted sample of configuration C45C45 during a fatigue tensile loading. In order to control that measure, the sample has been subject to RX tomography. The presence of tows/resin splittings and intra-tows crackings is observed. The parallel with the value of Sigma indicates that if the quality factor reaches its maximal value (in red on the figure) that corresponds to tows/resin splittings and if the software is unable to calculate its value (in grey on the figure), that corresponds to intra-tows crackings.

Configuration C45C45

For the configuration C45C45, the post-impact damage propagates at ±45 • until it reaches the edges of the sample (Figure 10). Figure 11 shows the value of the load measured during the test versus the number of cycles.

In can be divided in four steps (Figure 12) : The propagation initiates with tows/resin splittings from each tip of the initial fibres breakages. They progress transversely to the initial breakages in order to join into a unique point. Then, they propagate at ±45 • from the point of intersection and intra-tows crackings initiate. These two types of matrix damaging propagate at ±45 • but the intra-tows crackings propagation is much slower. Finally, the splittings reach the edges of the samples, following by the crackings some number of cycles later. No fibres breakages are visible by RX tomography after the fatigue loading (Figure 9). In fact, the damage propagation is governed by the resin. That explains why, for all the tested samples, when the damage propagation reaches the edges of the sample, the load is not equal to zero but is about 2.2 kN (Figure 11).

More, the increase of the impact energy or the displacement level leads to a decrease of the number of cycles necessary to initiate the damage propagation and a faster propagation of the damage (Figure 11 and Table 4).

However, these values converge with the increase of the impact energy.

In fact, from an energy of 4 J, the impactor perforates the laminates and the size of the impact damage is roughly the same (Figure 5). Finally, for the lower impact energy (∆L 2 ; 2.25 J) and displacement level (-20% ∆L 2 ; 4 J), the post-impact damage barely propagates (Figure 13).

The tests was stopped after 10 6 cycles. The load practically does not decrease during the fatigue test which is representative of few damage in the sample (Figure 11). The post-impact damage propagation also initiates with tows/resin splittings from each tip of the initial fibres breakages. However, they propagate both transversely and in the direction of the initial breakages.

Their progression is very slow and the intra-tows crackings initiate very late in comparison to the previous scenario (about 10 6 cycles). The propagation is not localized and several preferential areas of damage propagation are noticeable in this case (Figure 14). Hence, the samples does not crack on a ±45 • band.

Configuration C0C0

For the configuration C0C0, the post-impact damage propagates vertically (Figure 15). The DIC indicates the presence of vertical areas strongly loaded in shear (Figure 16). The damage propagates into this areas, where the principal strain directions are oriented at ±45 • . As for the configuration C45C45, the damage propagation is governed by the damage of the resin. In fact, the RX tomography, performed after the tests, shows the emergence of tows/resin splittings and intra-tows crackings (Figure 17). The progression in fatigue tension of tows/resin splittings and intra-tows crackings in the sample of configuration C0C0 is identical. The shape of their length curves is similar. Nevertheless, the splittings initiate sooner and faster than the intra-tow crackings.

More, the increase of the initial impact energy or the displacement level leads to a faster propagation of the post-impact damage and greatest final sizes of tows/resin splittings and intra-tows crackings. However, a gap is noticeable between (2.25 J ; 3 J) and (4 J ; 6.25 J ; 9 J). From an energy of 4 J, the impactor perforates the laminate. So, for the three higher energies, the initial impact damage size is roughly the same and the post-impact fatigue behaviour of the sample converges.

For the higher loading +25% ∆L 3 , the fracture surface (Figure 19) is identical to that observed for impacted samples subject to quasi-static tensile loading and described in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF]. Fibres breakages propagate in the direction perpendicular to the loading and the sample breaks almost instantly after the beginning of the fatigue loading (N failure = 50).

Configuration C45C0

For the configuration C45C0, the post-impact damage propagates vertically in the lower ply oriented at (0/90) • and propagates at ±45 • in the upper ply oriented at ±45 • (Figure 20). Contrary to C45C45 and C0C0, the observation of the damage indicates the development of delamination.

The damage propagation scenario is described in detail on the Figure 21.

Several samples have been subject to fatigue tensile loading. The tests have been stopped at 10 5 , 5.10 5 and 2.10 6 in order to analyze the damage propagation in the sample by RX tomography. The damage propagation begins in the lower ply oriented at (0/90) • : tows/resin splittings initiate and propagate vertically as observed for the configuration C0C0. That induces the initiation of delamination between the two carbon plies. Then, intra-tows crackings initiate and propagate vertically. However, unlike the configuration C0C0, the splittings progress vertically and also horizontally, as for the delamination. At the same time, in the ply oriented at ±45 • , intratows cracks propagates at ±45 • . Finally, all these damages propagate simultaneously during the fatigue loading. The resin damage in each ply is delimited by the delaminated area. This damage propagation scenario indicates that each ply of the configuration C45C0 behaves individually. It seems to be due to the delamination between the two plies which dissociates the specific behaviour of each orientation.

The influence of the impact energy on the damage propagation is observed on RX tomography performed after 2.10 6 cycles (Figure 22). The increase of the impact energy leads to a larger delaminated area and a more important matrix damage in the lower ply. Indeed, the curves of damage length depending on the number of cycles show a faster propagation of the tows/resin splittings and intra-tows crackings in the lower ply with the increase of the impact energy or the level of cycle level (Figure 23).

As for the configuration C45C45 and C0C0, the speed of damage propagation converges from an impact energy of 4 J for which the impactor perforates the laminates.

For the higher loading level +5% ∆L 4 , the sample breaks instantly after the beginning of the fatigue loading (Figure 24). Fibre breakages propagate between 0 • and ±45 • as for an impacted samples loaded with a quasi-static tensile loading, as shown in [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF].

Numerical study

Semi-continuous approach

In this part, the semi-continuous modelling strategy is extended to fatigue tensile simulation on carbon/epoxy woven laminates. It has been developed initially for impact loadings [START_REF] Navarro | Semi-continuous approach for the study of impacts on woven composite laminates: Modeling interlaminar behavior with a specific interface element[END_REF][START_REF] Pascal | On the modelling of low to medium velocity impact onto woven composite materials with a 2d semi-continuous approach[END_REF][START_REF] Pascal | Impact damage prediction in thin woven composite laminates -part i: Modeling strategy and validation[END_REF][START_REF] Pascal | Impact damage prediction in thin woven composite laminates -part ii: Application to normal and oblique impacts on sandwich structure[END_REF] and extended to post-impact quasi-static tensile loadings on woven laminates [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part ii: Numerical study[END_REF].

The modelling strategy is to decouple the resin and tows behaviour in order to represent simultaneously the damaged resin and the undamaged fibers tows (Figure 25). For this purpose, a specific finite element is developed. It relies on a truss structure of rod elements, modelling the woven fabric bundles and following the woven pattern geometry, and a shell element, representing the epoxy matrix. A specific interface element that have been developped in a previous work [START_REF] Pascal | On the modelling of low to medium velocity impact onto woven composite materials with a 2d semi-continuous approach[END_REF] , with an elastic damaging behaviour based on a bilinear cohesive law, connects the plies and represent the delamination.

The rod elements have an elastic and brittle behaviour. Their breakage are calculated through tensile and compressive strain criterions. The resin has an elastic and isotropic behaviour with an anisotropic damage in each direction of the woven through two damage variables, that are function of thermodynamical forces. It has also a pseudo-plastic behaviour in plane shear with a shear damaging law. It is calculated through two damage variables which represent respectively tows/resin splittings and cracks into bundles [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part ii: Numerical study[END_REF]. This original element formulation, so as the fatigue law presented in the next section, have been implemented in the explicit solver RADIOSS with the use of user FORTRAN subroutines in a 8-nodes "user element". a and Y 0 b have also been added for each damage type [START_REF] Miot | Rupture de structures composites stratifiées sous chargements statique et de fatigue[END_REF][START_REF] Hochard | A generalized damage model for woven ply laminates under static and fatigue loading conditions[END_REF]. These parameters are identified tests on the configurations C45C45 and C0C0.

Damage accumulation

The damage accumulation in the shell element during the fatigue tensile simulation is taken into account by a simple Miner's rule. A variable D m is introduced such as :

D m = i n i N i (2)
where n i is the number of cycles realized at an energy release rate Y i for which the number of cycles at failure is N i . Thus, if D m = 1, the the shell element is deleted.

Strategy of post-impact fatigue tensile simulation

In this section, the samples are modeled in the explicit solver RADIOSS with 8-nodes "user elements". The mesh size used for the modeling is equal to the physical pattern of the carbon woven which is 1,4mm. The number of elements is 20500 which corresponds to 30900 nodes.

Principle

The principle of the simulation strategy is to find in the model the shell element which requires the lowest number of cycles to reach the breakage.

That amounts to find the number of cycles N elem which verifies :

N elem = min j∈mesh (1 -D m (j)) * Y i Y j -Y 0 i 1 k (3)
with i = a or b and k = α or β.

Strategy

The strategy is implemented through a user law in the explicit finite element code Radioss. The user law is made up of a quasi-static and a fatigue module. The steps of the simulation are :

1. First a quasi-static rise is numerically performed until the maximum displacement of the experimental fatigue cycle : ∆L = ∆L sta + ∆L dyn .

A first calculation of strains and thermodynamical forces is done for each element of the model.

Then, the calculation goes into the fatigue module :

(a) The breakage number of cycles is calculated for each shell element of the model in order to find the first element to fail (Equ. 3).

(b) If the damage corresponds to : tows/resin splitting : the shell element does not have to be entirely deleted because the intra-tows crackings did not appear yet. The shear damage variable of this element is set to 0.5. intra-tows crackings : the shell element is entirely deleted ( the shear damage variable of this element is set to 1).

(c) Once the shell element is damaged, the number of cycles and all the variables D m (in order to simulate the damage accumulation) are uploaded.

3. Before moving to the next iteration, the calculation goes again into the quasi-static module in order to get the new strains and thermodynamical forces of each element of the model.

Fatigue tensile simulations on impacted carbon woven samples

Identification of the resin damaging laws

In this paragraph, the propagation laws presented in 3.2.1 are identified and validated through simulations on impacted samples of configuration C45C45 and C0C0. The impact energies and displacement levels used for these simulations are the same as in the experimental part (Table 3). For the configuration C45C45, the post-impact damage scenario given by the calculation is presented on the Figure 27. The damage propagates at ±45 • in the same way as the experimental scenario described in the paragraph 2.3.3.

Furthermore, the number of cycles required to reach the edges of samples obtained with the modelling are very close to the values experimentally obtained (Tables 56).

Figure 28 shows the energy release rates calculated at the boundary of the post-impact damage at the end of the quasi-static rise. The values are more important with the increase of the post-impact damage size or the displacement level. That explains why the damage propagates faster with the increase of the initial damage size or the displacement level (Tables 56).

More, as for the experimental testings, the post-impact damage barely propagates for the couples (∆L 2 ; 2.25 J) and (-20% ∆L 2 ; 4 J) (Figure 29).

The splittings progress very slowly and intra-tows crackings initiate very late (10 6 cycles). The simulation has been stopped at 2.10 6 cycles. This slow evolution can also be explained with the observation of the energy release rates calculated in the sample. For instance, for a low displacement level (-20 % ∆L 2 ), once the tows/resin start to propagate, these values are lower than the values obtained for a higher displacement level (Fig-ure 30). They are below the threshold value Y b 0 required to initiate intra-tows crackings. As a consequence, they do not emerge immediately and need a high number of cycles to appear.

Configuration C0C0.

For the configuration C0C0, the post-impact damage propagation scenario obtained with the modelling is in accordance with the experimental results presented in the paragraph 2.3.4. Tows/resin splittings and then intra-tows crackings propagate vertically (Figure 31). Furthermore, the post-impact damage propagation velocity well correlates the velocity experimentally obtained. The evolution curves depending on the number of cycles are presented on the Figure 32 for the tows/resin splittings and then intra-tows crackings.

They propagate very quickly in the 100 000 -200 000 first cycles before stabilizing progressively. In fact, shear strains in the resin at the tip of the damage propagation decrease as the simulation progresses (Figure 33). That leads to a decrease of the energy release rates in the resin. So, the number of cycles required to initiate splittings and crackings increases. Finally, for 10 7 cycles, energy release rates calculated at the tip of the splittings are lower to the damages threshold values Y a 0 and Y b 0 . With an increase of the initial damage size or the imposed displacement, the shear strains calculated at the tip of the post-impact damage at the end of the quasi-static rise increase. The energy release rates are higher and the resin damaging initiates faster (Figure 34). However, for the highest displacement level +30% ∆L 3 , fibres breakages propagate horizontally during the quasi-static rise before the start of the fatigue calculation (Figure 35). The fibres reach the tensile strain limit, so that the sample breaks as in quasi-static [START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part ii: Numerical study[END_REF].

Configuration C45C0

The semi-continuous modelling is validated for fatigue tensile simulations on carbon/epoxy woven laminates made up of two identical plies. However, a limitation is reached when the two plies have a different orientation. For instance, the Figure 36 shows the result of a simulation performed on a sample of configuration C45C0 initially impacted with an energy of 6.25 J and subjected to a displacement level ∆L 4 . The matrix damage in the two plies is not enough developed in comparison to what observed experimentally. This difference is due to the fact that delamination has not been implemented yet in the modelling. Indeed, in order to verify that delamination has to be accounted for, the strategy of post-impact fatigue tensile simulation presented in 3.3.2 has been modified. As for the shell elements, a simple Basquin-type fatigue propagation law is introduced for each interface element of the model. For this purpose, the number of cycles to failure is calculated through :

N f ailure interf ace = (1 -D m interf ace ) σ 6 σ II 1 λ (4)
where σ II is the mode II stress calculated in the cohesive interface, σ 6 is the failure stress at 10 6 cycles and λ is a parameter of the fatigue law. During the fatigue module of the strategy, the number of cycles to failure of the interface is also taken into account in order to find the element which requires the 20 lowest number of cycles to reach the failure.

The results are presented here in order to see if the delamination propagation in fatigue has a big influence on the phenomenon. Thus, the values for the parameters of the delamination fatigue law have not been identified very precisely. With this modified strategy, the simulation performed on the sample of configuration C45C0 gives now better results. Horizontal and vertical delaminated areas are observed during the simulation (Figure 37). The two carbon plies are locally debonded, so that strains and energy release rates in the C0 carbon plyare higher. That leads to bigger vertical damages in this ply. However, the matrix damage propagates only vertically in the C0 ply and is not as much developed in the C45 ply as experimentally.

Conclusion

The post-impact damage propagation in thin carbon/epoxy woven composite laminates under tensile fatigue loading has been investigated in this article. The first main contribution of this work is to provide a study of the influence of the initial damage size, through the variation of the impact energy, and the level of loading has been performed. A precise analysis of the damage propagation scenario has also been provided. The observation of the propagation has been performed with RX tomography and DIC. From this experimental study, significant results can be highlighted.

The damage propagation in fatigue in the configuration C45C45 and C0C0 is governed by the resin damaging. Two types of damaging have been identified : tows/resin splittings and intra-tows crackings. The splittings appear always before the crackings and these two damages evolve out of time.

For C45C45, the damage initiates from each tip of the initial damage in order to form a rhombus and then propagates at ±45 • until the edge of the sample.

But, if the initial damage size or the loading level is too small, the damage barely propagates. For C0C0, the resin damaging propagates vertically in the areas strongly loaded in shear. It quickly evolves in the 200 000 first cycles of fatigue before stabilizing due to the decrease of the resin strains at the tip of the propagation as the testing continues. However, if the imposed displacement is too high, the behaviour is identical to the one observed in quasi-static. Fibers breakages propagate horizontally very quickly at the beginning of the test. For the configuration C45C0, the post-impact damage propagation is a combination of the evolutions described above. First, resin damaging propagates vertically and horizontally in the carbon ply oriented at (0/90) • . Then, resin damaging propagates at ±45 • in the ply oriented at ±45 • . In fact, delamination dissociates the two plies : each ply behaves individually and is guided by the delamination.

The second main contribution is to provide a description of a modelling strategy that is able to represent the damage induced by an impact and also the propagation of this damage in fatigue loading with the same model. It

Figures 4 ,5 and 6 .

 46 Figures 4,5 and 6. The damage has only been characterized visually : as there are only two plies, the most important damages can be measured with this method. The values in yellow corresponds to the size of the damaged area in the upper ply and the values in white are the sizes of the fibres breakages in each ply.

2. 3 .

 3 Fatigue tensile tests on impacted samples 2.3.1. Characteristics of the fatigue tensile tests The fatigue tensile tests on impacted samples are conducted with an INSTRON machine with a load cell of 250 kN (Figure 7). They are controlled in displacement. The imposed cycle is presented on the Figure 8. It is typical of the loading of an helicopter blade. A quasi-static rise is imposed until ∆L sta and a cycle of half-amplitude ∆L dyn is imposed with a frequency of 15 Hz.

1 .

 1 A very high initial decrease explained in the literature by an important decrease of stiffness and a fast development of residual strains at the beginning of the test before stabilization [28] 2. A slower decrease due to the damage progression 3. A high diminution when the damage propagation reaches the edges of the sample 4. A stabilization for which the damage no longer progress

Figure 18

 18 Figure 18 shows the evolution of each type of damage versus the number of cycles. Damage 1 represents tows/resin splittings and Damage 2 represents intra-tows crackings.

3. 2 .

 2 Propagation laws in fatigue 3.2.1. Resin damaging The fatigue tensile tests, performed in 2.3, have shown that the postimpact damage propagation was governed by shear damaging with the development of tows/resin splittings and intra-tows crackings propagation in the laminates. These two types of resin damaging types follow the same direction and have a similar behaviour. However, the splittings initiate first and propagate faster than the crackings. As for the quasi-static modelling, the propagation in fatigue of resin damaging is based on thermodynamical forces. Two fatigue laws Y xy -N resin a and Y xy -N resin b are implemented for the shell element. N resin a and N resin b are the number of cycles necessary to initiate respectively the tows/resin splittings and the intra-tows crackings. They are expressed as Basquin curves : where Y a , α, Y b and β are parameters of the fatigue laws. Threshold values Y 0

  The propagation laws in fatigue Y xy -N cycles , identified for tows/resin splittings and intra-tows crackings, are presented on the Figure26.

3. 4 . 2 .

 42 Validation of the resin damaging laws Configuration C45C45.

  is innovative because the literature dealing with the modeling of the postimpact damage propagation in carbon/epoxy woven laminates subject to tensile loading is barely existing. In fact, most of the studies are conducted on laminates with notches, holes or pre-existing delamination. This original modeling strategy is developed at the mesoscopic scale. Indeed, the semi-continuous modelling has been extended to fatigue tensile simulations. The resin damage is implemented for the shell elements through two fatigue laws Y xy -N resin a and Y xy -N resin b based on thermodynamical forces. The damage accumulation is taken into account by a simple Miner's rule. Finally, the strategy is to find in the sample the shell element which requires the lowest number of cycles to reach the breakage and to delete it.The laws have been identified through several simulations on impacted samples of configuration C0C0 and C45C45. The modelling is validated in terms of damage propagation scenario, number of cycles at initiation and speed for woven carbon/epoxy laminates made up of plies with the same orientation. However, for the C45C0 configuration, it appears that delamination is a key phenomenon. Thus a fatigue law has to be properly implemented in order to represent the mechanisms leading to the post-impact fatigue behaviour of this configuration.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Experimental path diagram

Figure 4 :Figure 5 :Figure 6 :

 456 Figure 4: Post-impact fracture surfaces on C0C0 samples[START_REF] Rogani | Tensile post-impact behaviour of thin carbon/epoxy and glass/epoxy hybrid woven laminates -part i: Experimental study[END_REF]

Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 : 1 1e+05 1 -Figure 18 :Figure 19 :

 7891011121314151617111819 Figure 7: Fatigue tensile test on impacted sample

Figure 20 :Figure 21 :Figure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 27 :Figure 28 :Figure 29 :Figure 31 : 1 -

 202122232425272829311 Figure 20: Progression of the post-impact damage in a sample of configuration C45C0 subject to fatigue tensile loading

Figure 32 :Figure 34 :Figure 35 : 3 51Figure 36 :Figure 37 :

 32343533637 Figure 32: Length evolution of the tows/resin splittings (Damage 1) and the intra-tows crackings (Damage 2) depending on the number of cycles for the configuration C0C0 -Comparison numerical/experimental

L 2 =

 2 1.63 mm 1.47 mm + L 1 = 0,66 mm + 0,53 mm E 0.60 mm 0.54 mm + L 1 = 0,66 mm + 0,53 mm L 4 = 0.68 mm 0.61 mm + L 1 = 0,66 mm + 0

Table 3 :

 3 Impact energies and displacements used for the post-impact fatigue tensile tests

	55

Table 4 :

 4 Number of cycles necessary to initiate the damage propagation and reachthe edges of the sample depending on the impact energy and the displacement

	level
	56

Table 5 :

 5 Number of cycles necessary to reach the edges of the sample depending on the impact energy for the configuration C45C45 Displacement N edges EXP N edges NUM

	∆L 2	60 000	50 000
	+10% ∆L 2	15 000	12 000

Table 6 :

 6 Number of cycles necessary to reach the edges of the sample depending on the displacement level for the configuration C45C45 57

Acknowledgment

This work was granted access to the HPC resources of CALMIP supercomputing center under the allocation 2019-P09105.


Marianne Quéméneur
email: marianne.quemeneur@ird.fr

Sandrine Chifflet

Fourat Akrout

Amel Bellaaj-Zouari

Belhassen

Impact of cigarette butts on microbial diversity and dissolved trace metals in coastal 2 marine sediment 3 4

Keywords: cigarette butt, bacteria, diversity, marine sediments, trace metals, Mediterranean 39

Cigarette butts are the most common plastic form of litter found in the marine coast, threatening 22 the quality of the seawater and marine life. However, the impact of cigarette butts known to 23 contain toxic chemicals has been investigated to date in very few marine species. This study 24 aimed to evaluate the effects of cigarette filters (smoked or unsmoked) on the microbial 25 diversity inhabiting coastal sediments by high-throughput sequencing of the 16S rRNA genes.

26

Both bacterial structure and metals distribution were impacted by cigarette filter addition in 27 laboratory sediment experiments, compared to control sediment incubations without filter. Both 28 smoked and unsmoked cigarette filters decreased pH and dissolved Cd, Mo and V 29 concentrations in marine sediment incubations, while they increased dissolved Fe, Mn, Zn 30 levels in the surrounding environment. Smoked filters dramatically decreased the relative 31 abundance of the phyla Bacteroidetes and Cyanobacteria, while the members of the phyla 32 Gammaproteobacteria, Firmicutes and Thermotogae were enriched by smoked filters in marine 33 sediments. Bacterial taxa associated with deep marine environments or hydrothermal seep fields 34 were selected by smoked cigarette filters. This study demonstrated for the first time the 35 microbial community changes and impacts from toxic cigarette filters in coastal marine 36 sediments.

Introduction 42

Cigarette butts (CB) are one of the most common plastic forms of litter found in the 44 environment [START_REF] Kadir | Cigarette butts pollution and environmental impact-A 512 review[END_REF]. From 5 to 6 trillion cigarettes were smoked worldwide is in the northern coast of Sfax and corresponds to an urban beach intertidal area highly

Sample collection 124

Surficial sediments (0-5-cm layer) were sampled in October 2018 from one location 125 (34°46′05.2′′N-10°48′49.9′′E) at high tide and at 0.5 m from the shore with a plastic spatula 126 and distributed in sterile plastic bags (~1 kg). Seawater was collected using a polyethylene pre-127 cleaned bottle with HCl 10% v/v (analytical grade) then thoroughly rinsed with ultrapure water 128 (R = 18.2 MΩ.cm -1 ). Seawater and sediment samples were kept at 4°C until use (<3h after the ambient light and temperature). All experiments were performed in triplicates. At the beginning 148 of the experiments (T0), the seawater supernatant was filtered (0.22-µm) and stored in a metal-149 free polypropylene tubes at -20°C until chemical analyses (i.e. nutrients and metals). At the 150 end of the experiments (Tf), the cigarette filters (smoked or unsmoked) were removed from the 151 tubes and the seawater supernatant was filtered (0.22-µm) and stored in a metal-free 152 polypropylene tubes at -20°C until chemical analyses (i.e. nutrients and metals). The pH of 153 experiments was measured using a pH-meter (inoLab 7110, WTW) calibrated with three standard buffers (pH 4.0, 7.0 and 10.0 at 20 °C). Subsamples of sediment (T0 and Tf) were also is useful for reducing sample matrix content to about 0.2% of dissolved solids before entering 163 the plasma [START_REF] Field | Direct determination of 10 trace metals in 50 µL 495 samples of coastal seawater using desolvating micronebulization sector field ICP-MS[END_REF]. In AGD-ICP-MS analyses, the argon (Ar) gas flow through the 164 nebulizer is reduced while the total Ar gas flow to the plasma is maintained by the addition of 165 a make-up Ar gas flow to the aerosol leaving the spray chamber. The sample aerosol is thereby 166 diluted with Ar gas inside the ICP-MS sample introduction system. Analytical detection limits 167 were below the analysed samples. Accuracy of ICP-MS measurements were controlled using 168 certified reference nearshore seawater (CASS-6). Trace metals recoveries were between 98 and 169 108% except for Mn and Mo (78% and 122%, respectively; data not shown).

170 Inorganic nutrient concentrations were determined with a BRAN and LUEBBE Type 3 auto-171 analyzer according to standard methods (Tréguer and LeCorre, 1975). Dissolved Inorganic The oxidizing agents were sodium hydroxide and potassium persulfate for nitrogen and sulfuric 176 acid and potassium persulfate for phosphorus (Aminot and Kérouel, 2007). Gammaproteobacteria, Firmicutes and Thermotogae (Figure 2). A shift in relative abundance 277 of microbial families was also observed depending on experimental conditions (Figure 3).

Results and discussion

278

Both USF and SF conditions were dominated by Proteobacteria (40.2 ± 1.4% and 53.4 ± 2.7% 279 of total reads) and Firmicutes (21.5 ± 1.2% and 28.6 ± 2.3%; Figure 1). Among Proteobacteria, Cyanobacteria, Table S2), suggesting that other unmeasured environmental parameters (e.g.

314

organic compounds) could play a key role in shaping bacterial communities.

Dominant bacterial species in CB experiments

317

The number of abundant species (dominant OTU >1% of total reads) in sediment increased 318 over time in 96h incubations, but it was significantly lower in SF conditions (accounting for 319 77.0 ± 3.6%) than in NF controls (making up 42.0 ± 8.3%) at TF (p<0.05).

320

In USF conditions (unsmoked filter), dominant OTUs at TF were mainly affiliated to Firmicutes 321 (15.5 ± 8.7%) and Fusobacteria (12.7 ± 8.9%; Table S1). The majority of Firmicutes OTU 322 showed significant difference between treatments (p<0.05; Table S3). oil fields in USA [START_REF] Davey | Isolation of three 469 species of Geotoga and Petrotoga: two new genera, representing a new lineage in the bacterial 470 line of descent distantly related to the "Thermotogales[END_REF], while Arcobacter halophilus was isolated from 357 hypersaline lagoon water in the Hawaiian Islands [START_REF] Donachie | Arcobacter halophilus sp. nov., the 476 first obligate halophile in the genus Arcobacter[END_REF] and also found in 358 industrially multi-contaminated coastal sediment (Zouch et al., 2017).

359

No Cyanobacteria and Bacteroidetes, which are important in photosynthetic activities and 360 organic matter degradation, respectively, were represented among dominant species in 361 sediment incubations with CB (SF conditions, Table S2). The absence of such key microbial 362 groups among dominant bacteria in our experiments with smoked filters could be explained by 363 the release of toxic chemical compounds inhibiting their respective cell growth into surrounding 364 medium. However, Bacteroidetes species, particularly those belonging to the class 365 Flavobacteria, constitute a major component of the bacterial community in metal-and oil-contaminated marine sediment (Zouch et al., 2018;[START_REF] Kappell | The polycyclic aromatic hydrocarbon degradation potential of Gulf of Mexico 515 native coastal microbial communities after the Deepwater Horizon oil spill[END_REF]. In marine ecosystems,

367

Bacteroidetes were also known to growth attached to particles, surfaces or algal cells 368 (Fernandez-Gomez et al., 2013). Moreover, Cyanobacteria were also overrepresented on 369 plastic debris compared to the surrounding free-living and organic particle-attached fractions 370 in 'plastisphere' studies (Jacquin et al., 2019). Thus, the depletion of Cyanobacteria and 371

Bacteroidetes from sediment fraction might be also explained by: (i) the potential death of

Legends of Figures

413

Values are means of abundance data from biological triplicates ± standard deviation. The dendrogram was constructed from the family abundance table using Euclidean distance.

421

The scale represents 10% dissimilarity.

118

  frequented by local people. This coastal area had a lot of waste including CB and other litter 119 items (e.g. plastic bags and bottles, clothing, packaging) discarding by people or deposited by 120 coastal currents and tides, wind and rains, as observed in most urban coastal areas frequented 121 by people in Tunisia and other Mediterranean countries.122 123

  (Al, As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Sb, V, Zn) concentrations 159 were measured by Argon Gas Dilution -Inductively Coupled Plasma -Mass Spectrometry 160 (AGD-ICP-MS, iCAP-Q, Thermo Scientific) after samples acidification (ultra-pure HNO 3 1% 161 v/v). Because high dissolved solid contents induce ionization suppression, the AGD technique 162

172

  Nitrogen (DIN) is the sum of NO 2 -, NO 3 -and NH 4 + values. Dissolved Inorganic Phosphorus 173 (DIP) corresponds to PO 4 3 values. Dissolved Organic Nitrogen (DON) and Dissolved Organic 174 Phosphorus (DOP) were measured after mineralization processes at high temperature (120°C).

  175

  DNA extraction, PCR and sequencing of 16S rRNA gene fragmentsDNA extraction from triplicated initial sediment samples (without filter, NF-T0) and final 180 sediment samples (Tf) of the three experimental conditions (NF, USF, SF), performed in 181 biological triplicate (i.e., NF1, NF2, NF3; USF1, USF2, USF3; SF1, SF2, SF3), was carried out 182 using the UltraClean Soil DNA Isolation Kit (MoBio Laboratories, Inc., CA), as previously 183 described by[START_REF] Mei | Serpentinicella alkaliphila gen. nov., sp. nov., a novel alkaliphilic anaerobic 522 bacterium isolated from the serpentinite-hosted Prony hydrothermal field[END_REF]. Bacterial and archaeal 16S rRNA gene V4 variable 184 regions were amplified by PCR using the Pro341F/Pro805R prokaryotic universal primer set 185(Takahashi et al., 2014), with barcode on the forward primer, as previously described by Dowd 186 et al. (2008), and were sequenced by the MiSeq Illumina (paired-end 2 x 300 bp) platform of 187 the Molecular Research Laboratory (Texas, USA). Sequence data were processed using MR 188 DNA analysis pipeline (MR DNA, Shallowater, TX, USA). In summary, sequences were 189 joined, depleted of barcodes then sequences <150bp removed, sequences with ambiguous base 190 calls removed. Sequences were denoised, then Operational taxonomic units (OTUs) generated 191 and chimeras removed. OTUs were defined by clustering at 3% divergence (97% similarity). 192 Final OTUs were taxonomically classified using BLASTn against NCBI non-redundant (NR) 193 reference database. The 16S rRNA gene sequences of OTU have been deposited in the Genbank 194 database under the accession numbers MN463061-MN463096. results of a Shapiro-Wilk normality test, experimental data were analyzed 198 using the Analysis of Variance (ANOVA) followed by Tukey's post-hoc test or the non-199 parametric Kruskal-Wallis test followed by Dunn's test with Bonferroni correction to assess 200 the effect of treatments on the nutrients, pH and trace metals, and the relative abundance of 201 microbial taxa (classes/phyla and dominant phylotypes). P values of <0.05 were considered as 202 statistically significant differences. A Spearman rank correlation test was chosen to investigate 203 the relations among bacterial taxa and chemical parameters (showing significant differences 10 204 between treatments), and we accepted correlation coefficients (r S ) with P values of <0.05 as 205 significant associations. Linear regression analyses were run to assess relationships between 206 log-transformed response variables (bacterial classes/phyla showing significant differences 207 between treatments) and the most discriminant explanatory variables (metals and pH). The data 208 obtained at the beginning and at the end of the experiments were also analyzed by heatmap, 209 Hierarchical Clustering Analysis (HCA) and Principal Component Analysis (PCA). The most 210 discriminant variables (among metals, microbial taxa and pH) showing significant difference 211 between the experimental conditions (i.e. P values of <0.05) and explaining the sample 212 distribution on the PCA ordination were selected and represented on the biplot. All statistical 213 analyses were performed using XLSTAT 2019.1.2 (Microsoft Excel add-in program; 214 Addinsoft, Paris, France).

405Figure 1 .

 1 Figure 1. Composition of microbial communities at the phylum or class level in the sediment

Figure 2 .

 2 Figure 2. Comparison of the relative abundance of selected bacterial taxa (showing significant

Figure 3 .

 3 Figure 3. Heat map showing the relative abundance of the families in the sediment at the

Figure 4 .

 4 Figure 4. Principal Component Analysis (PCA) biplot showing the variation among the data

217 218 3.1. Characteristics of initial microbial ecosystem 219

		The initial coastal seawater used in laboratory experiments had a temperature of 20.9°C, a pH
	220	value of 8.3 ± 0.3, and variable levels of trace elements (Table 1). Nutrients were abundant with
	221	dissolved organic nitrogen (DON) and phosphorus (DOP) concentrations around 14.52 ± 1.72
	222	µM and 8.54 ± 2.81 µM, respectively. The initial microbial diversity of the sandy surficial
	223	sediment was dominated by five bacterial phyla: Proteobacteria (50.4 ± 3.3%), Bacteroidetes
	224	(19.0 ± 0.9%), Firmicutes (7.5 ± 0.4%), Cyanobacteria (4.7 ± 2.8%), Actinobacteria (4.5 ±
	225	1.3%; Figure 1). This bacterial community was diverse with only 5 abundant OTUs (>1%)
	226	belonging to classes Cyanophyceae, Gammaproteobacteria and Flavobacteriia (making up less
	227	than 10% of all reads). Archaea accounted for 7.5 ± 1.3% of the microbial community and was
	228	mainly represented by the family Halobacteriaceae (phylum Euryarchaeota; Figure 1).
	229
	230	3.2

. Variations of physicochemical parameters in CB experiments 231

		littered items in the environment and are potential sources of environmental pollution, including
	242	trace metals (Araújo and Costa, 2019; Dobaradaran et al., 2018; Novotny et al., 2011). In our
	243 268	experiments, different models of trace metals distribution were observed depending on cigarette
	244 269	filter addition (Table 1). Overall, dissolved trace metals in SF conditions were higher than USF 3.3. Microbial diversity in CB experiments
	275	of	six	bacterial	taxa:	Alphaproteobacteria,	Bacteroidetes,	Cyanobacteria,
	276
		The evolution of nutrients, pH and trace metals in seawater between the beginning (T0) and the
	232	end (Tf) of the 96h experiments are given in Table 1. No significant nutrient difference was
	233	observed between the studied conditions (p>0.05). Significant pH decreases were observed in
	234	both USF (unsmoked filter) and SF (smoked filter) conditions from 8.3 ± 0.3 (NF, T0) to 7.5 ±
	235	0.3 (p=0.007; USF, Tf) and 7.4 ± 0.1 (p=0.005; SF, Tf). On the contrary, no significant pH
	236	difference was observed between NF-T0 and NF-Tf controls (no filter; from 8.3 ± 0.3 at T0 to
	237	8.0 ± 0.3 at Tf; p>0.05), indicating that CB addition decreased the pH of seawater. The variation
	238	in environmental conditions, such as pH, alters the trace metals mobility between seawater and
	239	sediments by inducing changes in dissolved trace metals concentrations (Hamzeh et al., 2014)
	240	and variations in microbial responses (Zouch et al., 2018). CB are among the most numerous

245

conditions showing a higher release of trace metals from smoked than unsmoked CB. However 246 due to specific physicochemical conditions (pH, salinity) of coastal environment and chemical 247 behavior of trace metals, some elements (Al, As, Co, Cu, Ni, Pb and Sb) presented no significant 248 difference between NF, USF and SF conditions at Tf. These results showed a limited impact of 249 cigarette filters (smoked or unsmoked) on natural distribution of trace metals in this intertidal 250 environment. Conversely, both Fe and Mn levels significantly increased in seawater at Tf in 251 USF (277.13 ± 55.91 µg-Fe/L, p=0.01; 37.43 ± 4.14 µg-Mn/L, p=0.04) and SF conditions 252 (376.87 ± 176.04 µg-Fe/L, p=0.04; 37.46 ± 1.36 µg-Mn/L, p=0.01), compared to controls (4.53 253 ± 2.81 µg-Fe/L and 8.84 ± 5.65 µg-Mn/L, respectively). To a lesser extent, a similar pattern 254 was also observed for dissolved Zn. The increase in dissolved trace metals concentrations of 255 some elements (Fe, Mn and Zn) in both USF and SF conditions compared to NF conditions at 256 Tf, highlighted a trace metals inputs from cigarette filters in coastal environment. These results

257

agreed with

[START_REF] Moerman | Analysis of metals leached from smoked cigarette litter. 527[END_REF]

which showed that Fe, Mn and Zn in CB were highly 258 leached (10%, 23% and 19% respectively) in water. On the contrary, significant loss of 259 dissolved Cd was observed at Tf in both USF (0.010 ± 0.002 µg/L) and SF conditions (0.018 ± 260 0.005 µg/L) compared to NF controls (0.300 ± 0.103 µg/L; p<0.02). Similarly, high decrease 261 in dissolved V were measured at Tf in both USF (1.80 ± 0.15 µg/L) and SF conditions (2.02 ± 262 0.23 µg/L) compared to controls (6.31 ± 0.35 µg/L; p<0.02). To a lesser extent, a similar pattern 263 was also observed for dissolved Mo. The decrease in dissolved trace metals (Cd, Mo and V) 264 concentrations may be due to adsorption processes on CB. Indeed, cigarette filters (made of 265 cellulose acetate) are a synthetic polymer which may behave like other plastics exporting trace metals in the marine environment

(Ashton et al., 2010;[START_REF] Holmes | Adsorption of trace metals to plastic resin 505 pellets in the marine environment[END_REF] Holmes et al., 267 2014)

.

270

The microbial diversity in sediment according to USF conditions (unsmoked filter), SF 271 conditions (smoked filter) and NF controls (no filter) were evaluated at Tf (96h) and compared 272 to the initial microbial diversity in sediment (NF controls at T0). Changes in overall microbial 273 structure were observed at the phylum/class level depending on experimental conditions (Figure

274

1). Significant differences between treatments (p<0.05) were detected in the relative abundance

  They were grouped in 323 two families (Bacillaceae and Clostridiaceae) and related to fermentative Exiguobacterium, Oceanimonas spp., but Vibrio spp. (accounting for 13.5 ± 2.3% of SF reads) The other abundant and gammaproteobacterial phylotypes enriched by CB were closely 342 affiliated with Vibrio owensii (100% similarity with OTU647 and OTU1430), isolated from 343 diseased cultured crustaceans (Cano-Gomez et al., 2010), and Vibrio harveyi (98.9% similarity

	344	with OTU4626), a model marine and bioluminescent microorganism known as pathogen of
	345	aquatic fauna (Wang et al., 2015). Among Firmicutes, three OTU developed well in presence
	346	of CB and were affiliated to mesophilic and fermentative bacterial species belonging to genera
	347	Exiguobacterium (99.5% similarity), Alkaliphilus (92.7% similarity) and Serpentinicella
	348	(98.4% similarity). These species were isolated from deep South Africa gold mine and
	338	were only predominant in presence of CB. The abundant OTU11 was closely related to Vibrio

324

Alkaliphilus and Vallitalea species isolated from marine and/or hydrothermal ecosystems (Ben 325

Aissa et al., 2014; Ben Aissa et al., 2015;[START_REF] Kim | Exiguobacterium 518 aestuarii sp. nov. and Exiguobacterium marinum sp. nov., isolated from a tidal flat of the 519 Yellow Sea in Korea[END_REF]

. The dominant fusobacterial OTU2 326 was affiliated to the thermophilic and fermentative Hypnocyclicus thermotrophus isolated from 327 a microbial mat sampled near a hydrothermal vent in the Greenland Sea

[START_REF] Roalkvam | Hypnocyclicus 544 thermotrophus gen. nov., sp. nov. isolated from a microbial mat in a hydrothermal vent 545 field[END_REF]

. 328 Others dominant species were affiliated to Alphaproteobacteria (e.g. Roseibacterium genus) 329 and Gammaproteobacteria (e.g. Marichromatium genus), whose relative abundance increased 330 significantly with the addition of unsmoked filter (p<0.05). Unlike controls, Cyanobacteria 331 (particularly the families Chroococcales and Oscillatoriales) were not represented among 332 dominant species in USF conditions.

333

In SF conditions (smoked filter), abundant OTUs were mainly assigned to 334 Gammaproteobacteria, followed by Firmicutes, Alphaproteobacteria, Epsilonproteobacteria,

335

Halobacteria and Thermotogeae (Table

S4

). As detected in controls (without cigarette filter), 336 Gammaproteobacteria were dominated by Marinobacter and Marinobacterium spp., as well as 337 Idiomarina and 339 diabolicus (99.0% 16S rRNA sequence similarity), a mesophilic and polysaccharide-secreting 340 bacterium isolated from a deep-sea hydrothermal field in the East Pacific Rise (Raguénès et al., 341 1997). 349 serpentinite-hosted Prony hydrothermal field in New Caledonia (Takai et al., 2001; Ben Aissa 350 et al., 2015; Mei et al., 2016). Two others dominant phylotypes were only retrieved in presence 351 of CB and were affiliated to the classes Epsilonproteobacteria (Arcobacter halophilus, 99% 352 similarity) and Thermotogeae (family Petrotogaceae accounting for 8.6 ± 1.8% of SF reads 353 represented by Geotoga subterranea, 99.0% similarity with OTU5). The relative abundance of 354 this OTU5 increased significantly with the addition of smoked filter (p<0.05). The moderately 355 thermophilic and fermentative Geotoga subterranea, was isolated from brines collected from 356

  The changes in bacterial community diversity, and selection of specific groups by cigarette filter addition, support the 392 need to evaluate the effect of potential toxic substances on global bacterial diversity (e.g. 16S 393 rRNA PCR tests), in addition to test model marine bacterial species (e.g. A. fischeri

	394	bioluminescence inhibition test) in future development of ecotoxicology studies.
	395
	372	sensitive algal biomass and Cyanobacteria, causing hypoxia and providing competitive
	373	advantage for Firmicutes against Bacteroidetes in sediment, and/or (ii) a potential colonization Sustainable Development) in the framework of the French-Tunisian International Joint
	378
	379	4. Conclusions
	380
	381	This study showed that CB changed the microbial diversity of coastal marine sediment, and the

374

of CB by Cyanobacteria and Bacteroidetes. These latter assumptions remain to be verified 375 through future experiments analyzing both sediment and CB fractions, as previously 376 investigated in microbial colonization studies of plastic and microplastic

[START_REF] Harrison | Rapid bacterial colonization 500 of low-density polyethylene microplastics in coastal sediment microcosms[END_REF]

377

[START_REF] Dussud | Colonization 484 of non-biodegradable and biodegradable plastics by marine microorganisms[END_REF] Jacquin et al., 2019)

. 382 physicochemical parameters of its surrounding environment (e.g. pH, trace metals). Cigarette 383 filter addition decreased the pH of seawater and the concentrations of dissolved Cd, Mo and V, 384 whereas they increased dissolved Fe, Mn and Zn concentrations in coastal ecosystem. We also 385 found that smoked filter addition to coastal sediment led to a depletion in Cyanobacteria and 386 heterotrophic Bacteroidetes (especially Flavobacteriaceae and Saprospiraceae), with a 387 concomitant enrichment in heterotrophic Gammaproteobacteria (Alteromonadaceae and 388 Vibrionaceae), Firmicutes (Bacillaceae and Clostridiaceae), and Thermotogae 389 (Petrotogaceae) related to deep and/or hydrothermal ecosystems and adapted to extreme 390 conditions (e.g. high temperature and metals concentrations).

399 Laboratory "LMI COSYS-Med". The project leading to this publication has received funding 400 from European FEDER Fund under project 1166-39417. We thank L. Causse from the AETE-401 ISO platform (OSU/OREME, Université de Montpellier) for performing trace metals analysis 402 in seawater samples.

Table 1 . Chemical properties (pH) and dissolved parameters (nutrients and trace metals) measured at the beginning (T0) and at the end (Tf) of experiments without filter (NF, control), with unsmoked filter (USF) and smoked filter (SF).

 1 Values are means of data from biological triplicates ± standard deviation.

	Parameters	NF (T0)	NF (Tf)	USF (Tf)	SF (Tf)	Difference 1
	pH	8.3 ± 0.3	8.0 ± 0.3	7.5 ± 0.3	7.4 ± 0.1	NF > USF # SF
	Nutrients (µM)
	DIN 2	11.70 ± 0.70	9.89 ± 0.97	12.27 ± 1.54	11.51 ± 1.94
	DIP 3	0.77 ± 0.30	0.62 ± 0.42	0.22 ± 0.18	0.47 ± 0.37
	DON	14.51 ± 1.72	14.75 ± 1.69	17.57 ± 1.86	17.10 ± 1.15	NF # USF # SF
	DOP	8.54 ± 2.81	6.36 ± 3.64	2.95 ± 1.64	5.18 ± 3.47
	SiO 2	2.94 ± 0.50	2.26 ± 0.14	2.47 ± 0.51	2.88 ± 0.58
	Dissolved Metals (µg/L)
	Al	8.15 ± 2.61	8.14 ± 1.48	8.94 ± 0.35	9.93 ± 3.60
	As	3.33 ± 0.59	6.37 ± 1.50	6.43 ± 1.97	6.39 ± 1.25
	Co	0.10 ± 0.02	0.88 ± 0.27	0.55 ± 0.09	0.85 ± 0.19
	Cu	2.02 ± 2.96	6.14 ± 1.62	2.42 ± 2.46	4.98 ± 3.83	NF # USF # SF
	Ni	1.02 ± 0.95	2.36 ± 0.67	2.25 ± 0.85	3.57 ± 1.59
	Pb	0.04 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.07 ± 0.05
	Sb	0.40 ± 0.06	1.86 ± 0.17	1.83 ± 0.34	2.01 ± 0.13
	Cd	0.01 ± 0.00	0.30 ± 0.10	0.01 ± 0.00	0.02 ± 0.00
	Mo	10.25 ± 1.57	14.45 ± 0.17	10.52 ± 0.96	11.69 ± 0.81	NF > USF # SF
	V	1.85 ± 0.25	6.31 ± 0.35	1.80 ± 0.15	2.02 ± 0.23
	Fe	4.82 ± 0.43	4.53 ± 2.81	277.13 ± 55.91 376.87 ± 176.04
	Mn	9.77 ± 0.42	8.84 ± 5.65	37.43 ± 4.14	37.46 ± 1.36	NF < USF # SF
	Zn	4.06 ± 2.66	4.12 ± 1.54	6.22 ± 3.00	4.66 ± 0.89 3

Table S1 . Spearman's Rank correlation coefficients between the relative abundance of selected bacterial taxa and chemical parameters (showing significant difference between treatments) in sediment experiments.

 S1 Values in bold are significant at p 0.05. ≤

	Variables	Cd	Fe	Mn	Mo	V	Zn	pH
	Alphaproteobacteria	0,014 -0,713 -0,608 -0,133 -0,056 -0,385	0,503
	Bacteroidetes	0,245 -0,881 -0,776 0,000	0,091 -0,385	0,685
	Cyanobacteria	0,42	-0,916 -0,839 0,266	0,385 -0,266	0,650
	Gammaproteobacteria	0,476	0,287	0,133	0,294	0,189	0,224	-0,343
	Firmicutes	-0,294	0,72	0,783	0,168	0,140	0,231	-0,692
	Thermotogae	-0,189 0,867	0,783	0,196	0,077	0,224	-0,664

Table S2 . Regression coefficients between the relative abundance of bacterial taxa (response variables) and selected chemical parameters (explanatory variables) in sediment experiments.

 S2 Values in bold are significant at p 0.05. ≤

	Response variables	Predictors Coefficient	Standard error	t	Pr > |t|
		Constante	0,763	45,664	0,017	0,987
	Alphaproteobacteria	Fe Mn	-0,015 -0,014	0,015 0,203	-0,997 -0,069	0,352 0,947
		pH	1,926	5,504	0,350	0,737
		Constante	-4,235	74,735	-0,057	0,956
	Bacteroidetes	Fe Mn	-0,014 -0,189	0,024 0,333	-0,592 -0,568	0,573 0,588
		pH	3,313	9,007	0,368	0,724
		Constante	25,692	20,593	1,248	0,252
	Cyanobacteria	Fe Mn	-0,001 -0,236	0,007 0,092	-0,119 -2,568	0,909 0,037
		pH	-2,175	2,482	-0,876	0,410
		Constante	113,393	130,556	0,869	0,414
	Gammaproteobacteria	Fe Mn	0,041 -0,506	0,043 0,582	0,970 -0,870	0,364 0,413
		pH	-10,279	15,735	-0,653	0,534
		Constante	60,890	71,262	0,854	0,421
	Firmicutes	Fe Mn	-0,001 0,374	0,023 0,317	-0,023 1,178	0,982 0,277
		pH	-6,656	8,589	-0,775	0,464
		Constante	11,623	34,674	0,335	0,747
	Thermotogae	Fe Mn	0,017 -0,061	0,011 0,154	1,483 -0,392	0,182 0,707
		pH	-1,339	4,179	-0,320	0,758

Table S3 . Blast analysis on the dominant microbial species (OTUs >1% of total sequences) in sediment at the end of the experiments (Tf) with unsmoked filter (USF, in bold), compared to conditions with smoked filter (SF, Tf) or no filter (NF, controls, T0 and Tf).

 S3 Values are means of data from biological triplicates ± standard deviations.

	OTU name [NCBI Accession number]	Phylum/Class	NF (T0)	Relative abundance (%) NF (Tf) USF (Tf)	SF (Tf)	Taxonomy closest cultivates [NCBI Accession number]	Identity (%)
	OTU_8 [MN463083]*	Alphaproteobacteria	0.34 ± 0.03	2.14 ± 0.63	3.17 ± 0.79	0.46 ± 0.33 Roseibacterium beibuensis [MG383386]	100
	OTU_35 [MN463084]* Alphaproteobacteria	0.03 ± 0.01	0.08 ± 0.05	1.20 ± 0.20	0.08 ± 0.07 Rhodovulum marinum [AM696692]	99.53
	OTU_19 [MN463085]	Bacteroidetes	0.19 ± 0.04	0.98 ± 1.10	1.42 ± 1.13 0.01 ± 0.002 Phaeodactylibacter xiamenensis [NR_134132]	90.97
	OTU_30 [MN463086]	Bacteroidetes	0.02 ± 0.004	0.11 ± 0.11	0.69 ± 0.68	0.59 ± 0.50 Marinilabilia nitratireducens [NR_132609]	96.84
	OTU_16 [MN463087]	Balneolaeota	0.19 ± 0.03	1.76 ± 1.56	1.01 ± 0.33	0.05 ± 0.02 Gracilimonas halophila [NR_158001]	97.07
	OTU_50 [MN463088]	Deltaproteobacteria	0.02 ± 0.01	0.02 ± 0.02	0.63 ± 0.61	0.07 ± 0.10 Desulfovibrio psychrotolerans [NR_042581]	92.05
	OTU_60 [MN463089]	Epsilonproteobacteria	0.00 ± 0.01 0.01 ± 0.001	0.64 ± 0.66 0.01 ± 0.004 Sulfurovum lithotrophicum [CP011308]	88.24
	OTU_1 [MN463064]*	Firmicutes	0.43 ± 0.06	4.38 ± 1.67	4.33 ± 1.81	6.17 ± 1.22 Alkaliphilus transvaalensis [NR_024748]	92.69
	OTU_14 [MN463090]*	Firmicutes	0.04 ± 0.01	0.05 ± 0.01	3.36 ± 2.30	0.06 ± 0.01 Vallitalea pronyensis [NR_125677]	96.93
	OTU_21 [MN463091]*	Firmicutes	0.38 ± 0.04	0.19 ± 0.22	2.04 ± 0.29	0.33 ± 0.30 Pontibacillus litoralis [NR_116372]	84.48
	OTU_4 [MN463065]	Firmicutes	0.19 ± 0.01	0.16 ± 0.05	4.25 ± 4.52	5.60 ± 4.60 Pontibacillus litoralis [NR_116372]	84.00
	OTU_31 [MN463092]*	Firmicutes	0.08 ± 0.02	0.18 ± 0.20	0.82 ± 0.45	0.09 ± 0.08 Pontibacillus salicampi [MH283830]	87.28
	OTU_3 [MN463063]*	Firmicutes	0.10 ± 0.01	0.20 ± 0.15	0.61 ± 0.89	8.72 ± 6.31 Exiguobacterium aestuarii [MH881394]	99.55
	OTU_2 [MN463093]	Fusobacteria	0.11 ± 0.01	0.12 ± 0.03 12.68 ± 9.94	0.15 ± 0.05 Hypnocyclicus thermotrophus [NR_145867]	92.96
	OTU_22 [MN463094]* Gammaproteobacteria	0.03 ± 0.01	0.04 ± 0.01 10.88 ± 0.87	0.31 ± 0.26 Marichromatium gracile [LT991979]	99.55
	OTU_7 [MN463071]	Gammaproteobacteria	0.28 ± 0.04	3.18 ± 2.03	2.11 ± 0.85	3.54 ± 1.11 Marinobacter hydrocarbonoclasticus [MK131324]	100
	OTU_28 [MN463095]	Gammaproteobacteria	0.10 ± 0.02	0.30 ± 0.03	1.03 ± 0.78	0.07 ± 0.07 Alteromonas marina [MH746022]	99.55
	OTU_25 [MN463096]	Gammaproteobacteria	0.10 ± 0.02	2.06 ± 1.72	1.02 ± 0.85	0.09 ± 0.03 Idiomarina taiwanensis [KM407758]	99.55
	OTU_5 [MN463085]*	Thermotogae	0.09 ± 0.02	0.31 ± 0.36	0.98 ± 1.37	8.55 ± 1.88 Geotoga subterranea [NR_029145]	99.00
	* Significant differences between treatments using ANOVA or Kruskal-Wallis tests (P<0.05)

Table S4 . Blast analysis on the dominant microbial species (OTUs >1% of total sequences) in sediment at the end of the experiments (Tf) with smoked filter (SF, in bold), compared to conditions with unsmoked filter (USF, Tf ) or no filter (NF, controls, T0 and Tf).

 S4 Values are means of data from biological triplicates ± standard deviations. Significant differences between treatments using ANOVA or Kruskal-Wallis tests (P<0.05)

	OTU name [NCBI Accession n°]	Phylum/Class	NF (T0)	Relative abundance (%) NF (Tf) USF (Tf)	SF (Tf)	Taxonomy of closest cultivates [NCBI Accession n°]	Identity %
	OTU_12 [MN463061]	Alphaproteobacteria 0.06 ± 0.01 0.06 ± 0.01 0.16 ± 0.19 3.10 ± 2.76 Thalassospira australica [MH304396]	99.29
	OTU_13 [MN463062]	Epsilonproteobacteria 0.04 ± 0.01 0.04 ± 0.01 0.06 ± 0.03 3.05 ± 2.85 Arcobacter halophilus [MG195897]	99.53
	OTU_3 [MN463063]*	Firmicutes	0.10 ± 0.01 0.20 ± 0.15 0.61 ± 0.89 8.72 ± 6.31 Exiguobacterium aestuarii [MH881394]	99.55
	OTU_1 [MN463064]*	Firmicutes	0.43 ± 0.06 4.38 ± 1.67 4.33 ± 1.81 6.17 ± 1.22 Alkaliphilus transvaalensis [NR_024748]	92.69
	OTU_4 [MN463065]	Firmicutes	0.19 ± 0.01 0.16 ± 0.05 4.25 ± 4.52 5.60 ± 4.60 Pontibacillus litoralis [NR_116372]	80.00
	OTU_56 [MN463066]	Firmicutes	0.01 ± 0.00 0.02 ± 0.01 0.02 ± 0.01 0.61 ± 0.89 Serpentinicella alkaliphila [NR_152685]	98.35
	OTU_46 [MN463067]*	Firmicutes	0.01 ± 0.01 0.01 ± 0.00 0.05 ± 0.04 1.54 ± 0.87 Halolactibacillus miurensis [AB681280]	99.11
	OTU_36 [MN463068]	Firmicutes	0.12 ± 0.01 0.11 ± 0.04 0.14 ± 0.08 0.84 ± 0.68 Tissierella creatinini [NR_117155]	95.04
	OTU_9 [MN463069]	Gammaproteobacteria 0.09 ± 0.03 0.10 ± 0.03 0.35 ± 0.47 5.02 ± 8.47 Oceanimonas doudoroffii [NR_114185]	99.33
	OTU_10 [MN463070]	Gammaproteobacteria 0.14 ± 0.03 1.68 ± 2.18 0.45 ± 0.42 3.70 ± 2.55 Idiomarina seosinensis [MG575737]	100
	OTU_7 [MN463071]	Gammaproteobacteria 0.28 ± 0.04 3.18 ± 2.03 2.11 ± 0.85 3.54 ± 1.11 Marinobacter hydrocarbonoclasticus [MK131324]	100
	OTU_6 [MN463072]*	Gammaproteobacteria 0.20 ± 0.01 1.44 ± 0.72 0.76 ± 0.20 7.85 ± 3.27 Marinobacterium sediminicola [NR_044529]	100
	OTU_42 [MN463073]	Gammaproteobacteria 0.06 ± 0.01 0.06 ± 0.03 0.17 ± 0.10 1.02 ± 1.39 Marinobacter vinifirmus [KX418471]	99.55
	OTU_11 [MN463074]	Gammaproteobacteria 0.16 ± 0.00 0.26 ± 0.10 0.25 ± 0.11 5.35 ± 4.53 Vibrio diabolicus [MK308588]	100
	OTU_647 [MN463075]	Gammaproteobacteria 0.09 ± 0.01 0.12 ± 0.04 0.17 ± 0.08 2.69 ± 2.27 Vibrio owensii [CP033138]	100
	OTU_3745 [MN463076]	Gammaproteobacteria 0.03 ± 0.01 0.05 ± 0.02 0.05 ± 0.01 1.37 ± 1.39 Marinobacterium georgiense [MH044627]	98.88
	OTU_24 [MN463077]	Gammaproteobacteria 0.03 ± 0.01 0.06 ± 0.02 0.04 ± 0.02 1.34 ± 1.28 Marinobacterium georgiense [NR_114163]	98.66
	OTU_47 [MN463078]	Gammaproteobacteria 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.01 1.11 ± 1.20 Pseudomonas aestusnigri [MF155916]	99.78
	OTU_1430 [MN463079]	Gammaproteobacteria 0.05 ± 0.03 0.05 ± 0.02 0.07 ± 0.03 1.05 ± 0.89 Vibrio owensii [CP033137]	100
	OTU_4626 [MN463080]	Gammaproteobacteria 0.04 ± 0.01 0.05 ± 0.03 0.05 ± 0.02 0.91 ± 0.77 Vibrio harveyi [EU834007]	98.86
	OTU_5 [MN463081]*	Thermotogae	0.09 ± 0.02 0.31 ± 0.36 0.98 ± 1.37 8.55 ± 1.88 Geotoga subterranea [NR_029145]	100
	OTU_15 [MN463082]	Archaea/Halobacteria 0.75 ± 0.04 1.21 ± 0.83 0.59 ± 0.27 1.11 ± 0.38 Halogranum amylolyticum [NR_113451]	99.51
	*

Acknowledgments

397

This project was financially supported by IRD (French National Research Institute for

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Author statement MQ designed and performed experiments. FA, MQ and SC helped for data acquisition. AZ, MB, MQ and SC contributed to the analysis and interpretation of data. MQ wrote the manuscript in collaboration with SC. All authors read and commented on the draft manuscript. All authors agreed to the final version.