Ben Fida

Salem

Ben Said

Cristiana Cravo-Laureau

Ezzeddine Mahmoudi

Noëlle Bru

Mathilde Monperrus

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

Pr Robert Duran

Bacterial community assemblages in sediments under high anthropogenic pressure at Ichkeul Lake / Bizerte Lagoon hydrological system, Tunisia

Keywords: 16S rRNA sequencing, pollution, pesticide, PAH, network analysis

Evidence of bacterial

Introduction

Lagoons and lakes are highly productive ecosystems, providing important ecological services such as breeding grounds and nurseries for many fish and bird species [START_REF] Vasconcelos | Connectivity between estuaries and marine environment: Integrating metrics to assess estuarine nursery function[END_REF]. They are threatened by human activities, and therefore they need careful monitoring because they are very vulnerable [START_REF] Pérez-Domínguez | Current developments on fish-based indices to assess ecological-quality status of estuaries and lagoons[END_REF]. Their status may change rapidly in response to natural-and human-driven events (Cravo-Laureau et al., 2017a;Duran et al., 2015c). Sediments are complex habitats densely colonized by diverse microorganisms [START_REF] Brito | A case study of in situ oil contamination in a mangrove swamp (Rio De Janeiro, Brazil)[END_REF][START_REF] Chronopoulou | Impact of a simulated oil spill on benthic phototrophs and nitrogen-fixing bacteria in mudflat mesocosms[END_REF]Duran et al., 2015a;[START_REF] Fourcans | Molecular analysis of the spatio-temporal distribution of sulfate-reducing bacteria (SRB) in Camargue (France) hypersaline microbial mat[END_REF]. In sediments, microbial communities are involved in the decomposition of organic matter [START_REF] Schultz | Effects of bacterial dynamics on organic matter decomposition and nutrient release from sediments: A modeling study[END_REF]. Microorganisms play a key role in carbon, nitrogen and phosphorus cycling [START_REF] Andrews | Hot Spots and Hot Moments of Dissolved Organic Carbon Export and Soil Organic Carbon Storage in the Shale Hills Catchment[END_REF] as well as in determining the fate of pollutants (Bordenave et al., 2004a;Bordenave et al., 2004b;[START_REF] Guermouche M'rassi | Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons[END_REF][START_REF] Pringault | Temporal variations of microbial activity and diversity in marine tropical sediments (New Caledonia lagoon)[END_REF][START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF]. Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are considered as persistent organic pollutants (POPs), which are ubiquitous in aquatic ecosystems [START_REF] Doong | Characterization and composition of heavy metals and persistent organic pollutants in water and estuarine sediments from Gao-ping River, Taiwan[END_REF][START_REF] Kjellerup | Effects of activated carbon on reductive dechlorination of PCBs by organohalide respiring bacteria indigenous to sediments[END_REF][START_REF] Mwanamoki | Trace metals and persistent organic pollutants in sediments from river-reservoir systems in Democratic Republic of Congo (DRC): Spatial distribution and potential ecotoxicological effects[END_REF]. POPs are mainly derived from industrial and agricultural sources, as well as maritime traffic [START_REF] Chang | Concentration and fate of persistent organochlorine pesticides in estuarine sediments using headspace solid-phase microextraction[END_REF][START_REF] Wen | Linked Sediment/Contaminant Transport Model for Rivers with Application to the Buffalo River, New-York[END_REF].

They enter aquatic environments through atmospheric deposition and run-off [START_REF] Botta | Transfer of glyphosate and its degradate AMPA to surface waters through urban sewerage systems[END_REF][START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF]. Their fate in the marine environment depends on abiotic and biotic factors (for review, see [START_REF] Duran | Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment[END_REF]. The rapid development of molecular methods for microbial ecology has provided detailed information on microbial diversity and dynamics in marine sediments [START_REF] Bordenave | Diversity of ring-hydroxylating dioxygenases in pristine and oil contaminated microbial mats at genomic and transcriptomic levels[END_REF][START_REF] Stauffert | Response of archaeal communities to oil spill in bioturbated mudflat sediments[END_REF][START_REF] Terrisse | Variation of Oxygenation Conditions on a Hydrocarbonoclastic Microbial Community Reveals Alcanivorax and Cycloclasticus Ecotypes[END_REF][START_REF] Thiyagarajan | Temporal variation of coastal surface sediment bacterial communities along an environmental pollution gradient[END_REF]. The molecular methods, particularly high-throughput sequencing technologies have been proved to be useful tools for biomonitoring aquatic ecosystem (Cravo-Laureau and Duran, 2014;Cravo-Laureau et al., 2017b). Their utilization in combination with chemical analyses might shed light on the ecological role and the adaptation of bacteria to environmental factors. Correlation analyses allowed the exploration of the interactions between microorganisms and their habitat [START_REF] Barberan | Using network analysis to explore co-occurrence patterns in soil microbial communities[END_REF][START_REF] Fuhrman | Community structure of marine bacterioplankton: patterns, networks, and relationships to function[END_REF][START_REF] Llado | Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil[END_REF][START_REF] Williams | Demonstrating microbial co-occurrence pattern analyses within and between ecosystems[END_REF][START_REF] Yergeau | Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities[END_REF]. Particularly, such analyses have identified possible pollutant degraders [START_REF] Llado | Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil[END_REF][START_REF] Yergeau | Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities[END_REF], thereby obtaining valuable information for guiding the management of microbial communities to mitigate the effect of pollutants.

The Ichkeul Lake / Bizerte Lagoon hydrological system (Tunisia) is located in an area under high anthropic pressure. The Ichkeul Lake, Ramsar protected area, is important for migrating birds [START_REF] Ramdani | North African wetland lakes: Characterization of nine sites included in the CASSARINA Project[END_REF]. The contaminants entering into the Ichkeul Lake / Bizerte Lagoon hydrological system [START_REF] Stevenson | Palaeoecological and documentary records of recent environmental change in Garaet El Ichkeul: a seasonally saline lake in NW Tunisia[END_REF][START_REF] Trabelsi | Polycyclic aromatic hydrocarbons in superficial coastal sediments from Bizerte Lagoon, Tunisia[END_REF] have been shown to affect aquatic organisms and migratory birds [START_REF] Ben Said | Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon[END_REF][START_REF] Casagranda | A first quantification of the overall biomass, from phytoplankton to birds, of a Mediterranean brackish lagoon: Revisiting the ecosystem of Lake Ichkeul (Tunisia)[END_REF][START_REF] Gharbi-Bouraoui | Field study of metal concentrations and biomarker responses in the neogastropod, Murex trunculus, from Bizerta Lagoon (Tunisia)[END_REF]. During the past decades several projects have studied the PAHs in the sediment of Bizerte Lagoon and their impact on the benthic microorganisms and organisms [START_REF] Ben Said | Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon[END_REF][START_REF] Ben Said | Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia[END_REF][START_REF] Ben Said | Changes of benthic bacteria and meiofauna assemblages during bio-treatments of anthracene-contaminated sediments from Bizerta lagoon (Tunisia)[END_REF]Louati et al., 2013a;Louati et al., 2013b;[START_REF] Trabelsi | Polycyclic aromatic hydrocarbons in superficial coastal sediments from Bizerte Lagoon, Tunisia[END_REF].

Recently, we characterized the contamination of the Ichkeul Lake / Bizerte Lagoon hydrological system. Almost all stations exceeded the threshold effect level (TEL) of the standards published by NOAA [START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF]. The Bizerte Lagoon has been shown to contain industrial contaminants including hydrocarbon (PAHs, polycyclic aromatic hydrocarbons) and metallic compounds, while the Ichkeul Lake was found to contain agrochemicals contaminants including organochlorine pesticides such as endrin and dieldrin (Ben Salem et al., 2016a;Ben Salem et al., 2016b;[START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF]. In a previous study, based on 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) analysis investigating the bacterial communities in four contrasted stations of the Ichkeul Lake / Bizerte Lagoon hydrological system (two located at the Ichkeul Lake, and two at the Bizerte Lagoon), we demonstrated that bacterial community compositions were linked with the type and level of pesticide contamination at the dry season (Ben Salem et al., 2016a). The current study aims to characterize the bacterial communities from the sediments of the Ichkeul Lake / Bizerte Lagoon hydrological system. We further characterize the bacterial communities structure by 16S rRNA genes T-RFLP analysis, expanding the study by investigating 20 stations around the Ichkeul Lake / Bizerte Lagoon hydrological system at two contrasted seasons (April, dry season; September, wet season). Also, we further characterize the bacterial composition of four contrasted stations by sequencing 16S rRNA gene PCRamplified fragments via 454-pyrosequencing. The effect of organic contaminants such as PAHs, PCBs, OCPs on the bacterial assemblages were determined. Specific bacteria, which abundance was linked with the presence of a pollutant, were identified.

Materials and Methods

Site description

The Ichkeul Lake / Bizerte Lagoon hydrological system, North Tunisia, suffers the effects of human activities because it is located in an area under high anthropic pressure. Bizerte Lagoon has a central role because connected to the Mediterranean Sea and to the Ichkeul Lake via the Tinja River; the hydrological system receives water via different rivers (Oueds) all around (Fig. 1). The lake experiences large fluctuations in salinity each year, from freshwater with average salinity around 4 PSU (Practical salinity unit) in winter (wet season) due to rivers flood, to saline ecosystem with average salinity around 35 PSU in summer (dry season) receiving seawater from the Bizerte Lagoon and because of evaporation. In a previous study, we demonstrated that pollution levels fluctuated according to seasons (Ben Salem et al., 2016a).

Sampling

Sediment samples were collected from 20 stations (ST) located all around the Ichkeul Lake / Bizerte Lagoon hydrological system (Fig. 1): ST1 to ST8 in the lake and ST11 to ST20 in the lagoon, ST9 on the channel connecting the lagoon to the sea, and ST10 in the Mediterranean Sea. The lake and lagoon stations were located at the river mouths (13 stations), close to industrial and agricultural areas (4 stations), and at the centers of the lake and the lagoon (2 stations). Two sampling campaigns were performed: September 2011 (wet season) and April 2012 (dry season). Surface sediments (0-10 cm) were collected in triplicates (3 independent samples, i.e. biological replicates) at each station with a Van Veen grab as previously described (Ben Salem et al., 2016a). Collected sediments were dispatched in cryotubes (3 technical replicates were taken), quickly frozen in liquid nitrogen for microbial analyses, and in glass bottles kept at 4°C for chemical analyses. At lab, samples for microbial analyses were stored at -80°C and those for chemical analyses at -20°C until use.

Physical-chemical analyses

Physical-chemical parameters, including temperature, dissolved oxygen, pH, salinity, phosphate, ammonia, nitrite, and nitrate were determined as previously described (Ben Salem et al., 2016a). Chemical analyses for contaminants followed the QuEChERS procedure described in Ben Salem et al. (2016b). Chemical data and physical-chemical parameters are summarized in supplementary material (Table S1). All chemical data and physical-chemical parameters are presented in details in Ben [START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF].

The contamination contents of the sediments were compared to sediment quality guidelines (SQG) in order to determine their potential toxic effect. Sediments were classified according the quality criteria developed by [START_REF] Bakke | Development of sediment quality criteria in Norway[END_REF] for PCBs, OCPs, and PAHs. The toxic potential of the sediments was evaluated by calculating the mean effect range medium (ERM) quotient (m-ERM-Q) as previously described [START_REF] Long | Predicting toxicity in marine sediments with numerical sediment quality guidelines[END_REF].

DNA extraction, PCR and T-RFLP analyses

DNA extractions were performed using 250 mg of frozen sediment with the UltraClean soil DNA isolation Kit (MoBio Laboratories, CA) as previously described [START_REF] Precigou | Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase[END_REF].

The bacterial universal primers 1387R 5'-GGGCGGWGTGTACAAGGC-3' and 63F 5'-CAGGCCTAACACATGCAAGTC-3' [START_REF] Marchesi | Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[END_REF] were used for 16S rRNA gene amplifications. Primers were fluorescently labeled for T-RFLP analysis, which was performed as previously described [START_REF] Precigou | Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction[END_REF] using 10 ng of extracted DNA. PCR mixture and conditions were performed as previously described [START_REF] Vercraene-Eairmal | Diuron biotransformation and its effects on biofilm bacterial community structure[END_REF]. PCR amplicon purification, digestion and electrophoresis separation have been previously described [START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oilcontaminated sludge[END_REF]. T-RFLP profiles were analyzed as previously described [START_REF] Cravo-Laureau | Role of environmental fluctuations and microbial diversity in degradation of hydrocarbons in contaminated sludge[END_REF].

Sequencing and data analysis

Among the 20 stations, 4 stations were selected according to the environmental parameters for 454 pyrosequencing analyses. The analysis was performed with the samples collected at the dry season (April 2012) because the highest levels of contaminants (particularly PAHs and pesticides) were recorded (supplementary material Table S1), and also because important contrasted conditions were detected between Ichkeul Lake and Bizerte Lagoon (Table 1, and supplementary material, Table S1). Two stations from the Ichkeul Lake (salinity 2.6 ± 0, pH 8.29 ± 0.02; supplementary material Table S1) were chosen for contrasted contaminations: ST5, showing multiple contaminations by PAHs and pesticides; and ST8, the most contaminated by OCPs (Table 1; supplementary material, Table S1). Similarly, two stations from the Bizerte Lagoon (salinity 29.5 ± 0.4, pH 8.37 ± 0.03) were chosen for contrasted contaminations: ST12, the most contaminated by PAHs; and ST17, the less contaminated of the entire complex (Table 1; supplementary material, Table S1). Three replicates for each station were sequenced at Molecular Research LP DNA (MR DNA sequencing service, Shallowater, TX, USA) using Roche 454 FLX titanium as previously described [START_REF] Dowd | Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)[END_REF]Duran et al., 2015a). QIIME (Quantitative Insights Into Microbial Ecology; [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF] was used to analyse the 16S rRNA gene sequence reads as described [START_REF] Terrisse | Variation of Oxygenation Conditions on a Hydrocarbonoclastic Microbial Community Reveals Alcanivorax and Cycloclasticus Ecotypes[END_REF]. Briefly, data cleaning was performed according to [START_REF] Quince | Accurate determination of microbial diversity from 454 pyrosequencing data[END_REF] by denoising of the data and by eliminating chimera and sequences with length under 450 bp, with mistakes in primers sequences or homopolymers. OTU picking was performed following the Usearch method [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF] defining reference sequences by applying a 97% similarity threshold. The taxonomic assignment was performed with BLASTn against May 2013 Greengenes database [START_REF] Desantis | Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB[END_REF][START_REF] Mcdonald | An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea[END_REF] to establish the OTU table. The OTU abundances for each sample were rarefied to the minimum read number observed (12,977 sequences) for a sample using the R package vegan [START_REF] Oksanen | Vegan: Community Ecology Package[END_REF]; Version: 2.0-10).

The complete dataset was deposited in the NCBI Sequence Read Archive (SRA) database (SUB5043721). It is available under the Bioproject ID PRJNA515186.

Statistical analysis

Canonical Correspondence Analysis (CCA) correlating bacterial community structure (T-RFLP patterns) with chemical data (PAH and pesticide concentrations) and physical-chemical parameters, was performed with MVSP v3.22 software (Kovach Computing Service, Anglesey Wales). T-RFLP profiles were normalized by calculating relative abundances of each TRF, as previously described [START_REF] Dominique | Simulation of the chemical fate and bioavailability of liquid elemental mercury drops from gold mining in Amazonian freshwater systems[END_REF]. Data were square root transformed to get a normal distribution of the data [START_REF] Dollhopf | Interpreting 16S rDNA T-RFLP data: Application of self-organizing maps and principal component analysis to describe community dynamics and convergence[END_REF].

All statistical analyses based on sequencing data were performed on the random subsample of 12,977 bacterial sequences, corresponding to the smaller number of sequences per sample in the dataset. Alpha diversity indices (Shannon-Wiener H' and richness R), based on rarefied OTU table, were calculated using PRIMER v5.0 (Plymouth Routines in Multivariate Ecological Research). The overall differences of these indices between the stations were tested by ANOVA and Tukey HSD multiple comparisons (pairwise comparisons).

Significant difference was assumed when p < 0.05. The contribution of individual OTUs explaining the differences between stations was determined by SIMPER (similarity percentages) based on Bray-Curtis distance measure.

Linear discriminant analysis effect size (LEfSe; [START_REF] Segata | Metagenomic biomarker discovery and explanation[END_REF] was performed to determine biomarkers of contamination and habitat as previously described (Jeanbille et al., 2016b). Briefly, the non-parametric Kruskal-Wallis sum-rank test (α = 0.05) was performed to detect taxa with significant differential abundance. The biological consistency was investigated by performing a pairwise Wilcoxon test (α = 0.05). A LDA threshold score of 2.0 and 1000 bootstrap interations were applied. Heatmap and clustered correlation [START_REF] Weinstein | An information-intensive approach to the molecular pharmacology of cancer[END_REF] were performed with CIMminer (http://discover.nci.nih.gov/cimminer/). These analyses were performed at the genus level derived from the OTU abundance table in order to determine the correlations between bacterial genera abundances and pollutant concentrations. Correlation analysis was based on Pearson correlation. The hierarchical clustering of correlations was based on ward method.

Strong correlations (>|0.5|) were considered. The distribution of OTUs identified in the bacterial patterns of the different stations was visualized with Venn diagrams constructed with Venny using OTU occurrence table as previously described (Duran et al., 2015a). OTUs present at least in two of the triplicates were retained for the analysis.

Results and discussions

Contamination level

The chemical analyses of the sediments collected during September 2011 and April 2012 sampling campaigns (supplementary material, Table S1) have been presented in detail in Ben [START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF]. endrin, dieldrin and lindane, and their derivatives, were detected in the Ichkeul Lake at concentrations ranging from 160 ± 14 to 2021 ± 85 ng g -1 . Additionally, the chemical analyses revealed low PCB contamination of the sediments (ranging from 2.48 ± 0.8 to 5.58 ± 1 ng g -1 ). The sediments were classified as class I (insignificantly polluted) except for the station ST8 that was classified as class II (moderately polluted). In order to take into account the multiple contaminations, the mean effect range medium quotients (m-ERM-Q) were calculated (Table 1; supplementary material, Table S1). The m-ERM-Q showed that the toxic potential of the sediments was higher (p value <0.01) in Ichkeul Lake stations than in Bizerte Lagoon stations at both seasons (supplementary material, Table S1). The station were classified according to increasing m-ERM-Q potential toxicity of sediments as follow ST17 < ST12 < ST5 < ST8 (Table 1). Station ST17 was considered as lowly contaminated, while stations ST5, ST8 and ST12 were considered as highly contaminated.

The chemical data and physical-chemical parameters (supplementary material, Table S1) presented in detail in Ben [START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF] were used in the present study to characterize the bacterial community structure according to environmental parameters.

Bacterial community structure

Correspondence canonical analysis (CCA) using T-RFLP patterns and chemical data separated bacterial communities of the two ecosystems, lake and lagoon irrespective of the season (Fig. 2a). Regarding the environmental parameters, the salinity, the temperature, and the concentrations of nitrite and ammonia were the main factors influencing the bacterial community composition (based on T-RF abundances) of the lagoon, while the bacterial community composition of the lake correlated with dissolved oxygen (DO) and phosphate concentrations. This observation was consistent with previous studies showing that bacterial communities are affected by salinity (Jeanbille et al., 2016a;Jeanbille et al., 2016b;[START_REF] Xi | Salinity influence on soil microbial respiration rate of wetland in the Yangtze River estuary through changing microbial community[END_REF] and nutrients [START_REF] Bodoque | Recovering hydromorphological functionality to improve natural purification capacity of a highly human-modified wetland[END_REF][START_REF] Liu | pH levels drive bacterial community structure in sediments of the Qiantang River as determined by 454 pyrosequencing[END_REF][START_REF] Yao | Biodiversity and ecosystem purification service in an alluvial wetland[END_REF]. Particularly, nitrite and ammonia concentrations may affect the bacterial populations involved in the nitrogen cycle in lagoon sediments as shown for saline lake sediments [START_REF] Lipsewers | Abundance and Diversity of Denitrifying and Anammox Bacteria in Seasonally Hypoxic and Sulfidic Sediments of the Saline Lake Grevelingen[END_REF] and marine contaminated sediments [START_REF] Mahmoudi | Microbial community composition and diversity in Caspian Sea sediments[END_REF]. The bacterial populations involved in the nitrogen cycle have been shown to play an important role in hydrocarbon removal in marine sediments [START_REF] Mahmoudi | Microbial community composition and diversity in Caspian Sea sediments[END_REF][START_REF] Stauffert | Structure of hydrocarbonoclastic nitratereducing bacterial communities in bioturbated coastal marine sediments[END_REF]. The CCA also showed that the pH and the concentrations of nitrate and organo-tin compounds influenced slightly the bacterial community compositions probably because they varied within a small range in the studied sediments.

Regarding the contaminants, the CCA analysis showed that the bacterial community composition was correlated with pesticide concentrations in the lake, while bacterial community composition was linked with PAHs concentrations in the lagoon (Fig. 2a). These observations confirmed those previously reported [START_REF] Ben Said | Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon[END_REF] that showed the impact of PAHs on bacteria communities in Bizerte Lagoon sediments. Considering the lake, the bacterial community composition was correlated with PAHs concentrations in April, except for those of station ST8 that was correlated with PCBs and Pesticides concentrations, while bacterial community composition was correlated with the concentration of organo-tin compounds in September (Fig. 2b). This observation suggested seasonal fluctuations in the input of contaminants. It is likely that the lake receives pesticide inputs from crop treatments in April. Additionally, the hydrology of the ecosystem might also play a crucial role: during the dry season (April) lagoon water contaminated by PAHs enters into the lake (Ben [START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF]. Considering the lagoon, the bacterial community composition was correlated with

PAHs and PCBs concentrations in April (Fig. 2c), suggesting that the pollutants were concentrated (concentrating effect) during the dry season. It is likely that the dry season represent a critical season for the Ichkeul Lake / Bizerte Lagoon hydrological system because the environmental conditions favor the concentrating effect of pollutants in both the lake and the lagoon. It is thus of paramount importance to investigate how benthic bacterial communities are affected by such inputs of pollutants. The sediments from the stations ST5 and ST8 (lake) and the stations ST12 and ST17 (lagoon) collected in April were retained for further bacterial characterization because they presented different type of contamination while slightly influenced by environmental parameters (Fig. 2b,c; Table 1).

Bacterial community composition

In order to further investigate the effect of pollutants on bacterial communities at the dry season (April), the composition of bacterial communities was determined by high throughput pyrosequencing. Four stations showing contrasted chemical characteristics were chosen (Table 1): station ST17 (lagoon), the less contaminated; station ST12 (lagoon), the most contaminated with PAHs; station ST8 (lake), the most contaminated with OCPs (pesticides);

and station ST5 (lake), contaminated with PAHs and OCPs mixture. Three replicates from each station were analysed. After trimming, the 191,278 sequences (400 bp) retained (12,977 sequences per sample after resampling) were dispatched within 858 OTUs, which were grouped into 44 different phyla (Fig. 3). Rarefaction curves (Supplementary data Fig. S1)

showed high sequencing coverage in all samples. The coverage (~ 94%) indicated that the sequencing effort was sufficient to assess the bacterial community diversity (Table 2). The sequence affiliation showed that Proteobacteria largely dominated the sediments, with predominance of Deltaproteobacteria (24%), Gammaproteobacteria (23%) and Alphaproteobacteria (9%); Epsilonproteobacteria and Betaproteobacteria accounting for 6% and 5% respectively. The dominance of Proteobacteria, particularly Deltaproteobacteria and Gammaproteobacteria, has been often reported in both lake and lagoon sediments [START_REF] Borin | Biodiversity of prokaryotic communities in sediments of different sub-basins of the Venice lagoon[END_REF][START_REF] Obi | Microbial Communities in Sediments of Lagos Lagoon, Nigeria: Elucidation of Community Structure and Potential Impacts of Contamination by Municipal and Industrial Wastes[END_REF][START_REF] Pavloudi | Salinity is the major factor influencing the sediment bacterial communities in a Mediterranean lagoonal complex (Amvrakikos Gulf, Ionian Sea)[END_REF][START_REF] Quero | Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)[END_REF][START_REF] Sun | Core sediment bacteria drive community response to anthropogenic contamination over multiple environmental gradients[END_REF] suggesting that they have a pivotal role in biogeochemical cycles in sediment environments.

Besides Proteobacteria, Bacteroidetes (9%), Chloroflexi (6%), Planctomycetes (2%) and Nitrospirae (2%) were the major phyla detected in all four stations (Fig. 3), which was consistent with previous observation in sediment environments [START_REF] Behera | Salinity and macrophyte drive the biogeography of the sedimentary bacterial communities in a brackish water tropical coastal lagoon[END_REF][START_REF] Fan | Characterizing bacterial communities in tilapia pond surface sediment and their responses to pond differences and temporal variations[END_REF]. These phyla have been detected in freshwater and marine sediments [START_REF] Behera | Salinity and macrophyte drive the biogeography of the sedimentary bacterial communities in a brackish water tropical coastal lagoon[END_REF][START_REF] Fan | Characterizing bacterial communities in tilapia pond surface sediment and their responses to pond differences and temporal variations[END_REF][START_REF] Jacquiod | Long-term soil metal exposure impaired temporal variation in microbial metatranscriptomes and enriched active phages[END_REF][START_REF] Paissé | Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution[END_REF][START_REF] Pavloudi | Salinity is the major factor influencing the sediment bacterial communities in a Mediterranean lagoonal complex (Amvrakikos Gulf, Ionian Sea)[END_REF].

Bacteroidetes are well known for their capacity to degrade organic matter [START_REF] Hunter | Microbial community diversity associated with carbon and nitrogen cycling in permeable shelf sediments[END_REF][START_REF] Zeng | Phylogenetic diversity of sediment bacteria in the northern Bering Sea[END_REF]. They were in high abundance in contaminated sediments [START_REF] Paisse | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Quero | Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)[END_REF][START_REF] Sun | Core sediment bacteria drive community response to anthropogenic contamination over multiple environmental gradients[END_REF]. Chlorofexi have been found as main players in the transformation of organic compounds including organo-chlorines such as PCBs and pesticides [START_REF] Fagervold | Microbial reductive dechlorination of Aroclor 1260 in Baltimore Harbor sediment microcosms is catalyzed by three phylotypes within the Phylum chloroflexi[END_REF][START_REF] Hug | Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling[END_REF]Matturro et al., 2016a). Planctomycetes, which have the capability to oxidize ammonia aerobically and anaerobically [START_REF] Junier | Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment[END_REF], have been widely detected in contaminated sediments [START_REF] Dell'anno | High bacterial biodiversity increases degradation performance of hydrocarbons during bioremediation of contaminated harbor marine sediments[END_REF][START_REF] Quero | Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)[END_REF].

Similarly, Nitrospirae members playing an important role in the nitrogen cycle [START_REF] Daims | The family nitrospiraceae, The Prokaryotes: Other Major Lineages of Bacteria and The Archaea[END_REF] have been detected in contaminated sediments by organic compounds including pesticides and PAHs [START_REF] Ding | Impact of pyrene and cadmium co-contamination on prokaryotic community in coastal sediment microcosms[END_REF][START_REF] Sánchez-Soto | Assessment of the bacterial community structure in shallow and deep sediments of the Perdido Fold Belt region in the Gulf of Mexico[END_REF][START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF].

Alpha diversity indices were not significantly different between stations (Table 2), an observation that was in contradiction with other studies showing that the presence of pollutants either reduce [START_REF] Haller | Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (Lake Geneva, Switzerland)[END_REF][START_REF] Johnston | Contaminants reduce the richness and evenness of marine communities: a review and meta-analysis[END_REF] or increase (Duran et al., 2015a;Jacquiod et al., 2018a) bacterial diversity. It was thus surprising to observe similar bacterial diversity indexes in sediments presenting different pollutant types and levels. Similar observations have been reported by comparing uncontaminated and contaminated sediments [START_REF] Paisse | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Yin | An integrated insight into the response of sedimentary microbial communities to heavy metal contamination[END_REF] and soils [START_REF] Berg | Selection for Cu-tolerant bacterial communities with altered composition, but unaltered richness, via long-term cu exposure[END_REF]. The pollution history, particularly the fluctuant and successive pollution loads resulting in legacy effects, may explain the fact that bacterial diversity was maintained [START_REF] Jurburg | Legacy Effects on the Recovery of Soil Bacterial Communities from Extreme Temperature Perturbation[END_REF][START_REF] Nunes | Soil bacteria show different tolerance ranges to an unprecedented disturbance[END_REF]. Also, the coalescence of bacterial communities, mixing bacterial communities from different ecosystems [START_REF] Mansour | Application of the microbial community coalescence concept to riverine networks[END_REF][START_REF] Rillig | Interchange of entire communities: Microbial community coalescence[END_REF], here lake and lagoon, may contribute to attenuate the diversity differences between the ecosystem, as previously described in river sediments [START_REF] Yin | An integrated insight into the response of sedimentary microbial communities to heavy metal contamination[END_REF]. The Venn diagram, showing shared OTUs between lake and lagoon stations (Fig. 4a) evidenced the ecological continuum between the lake and the lagoon, which are closely linked via the Tinja River (Fig. 1). Although similar diversity indexes were observed, the composition of the bacterial diversity was different.

Indeed, the Venn diagram revealed that each station was characterized by specific OTUs, only 211 OTUs being shared between the stations (Fig. 4a). Such observation was consistent with previous reports on bacterial communities in coastal areas (Duran et al., 2015b;[START_REF] Ikenaga | Changes in Community Structure of Sediment Bacteria Along the Florida Coastal Everglades Marsh-Mangrove-Seagrass Salinity Gradient[END_REF][START_REF] Misson | Chemical multi-contamination drives benthic prokaryotic diversity in the anthropized Toulon Bay[END_REF], indicating that bacterial communities were structured according to the environmental conditions, as revealed by the T-RFLP analyses (Fig. 2). Additionally, linear discriminant analysis effect size (LEfSe) detected taxa differentially abundant according the ecosystems. LEfSe showed the habitat specificity of genera that were specifically more abundant in either the lake or the lagoon (Fig. 4b). This observation was consistent with previous reports showing habitat specificity for microbial taxa (Jeanbille et al., 2016a;Jeanbille et al., 2016b;[START_REF] Xi | Salinity influence on soil microbial respiration rate of wetland in the Yangtze River estuary through changing microbial community[END_REF]. Similarly, LEfSe revealed the presence of specific genera (Sulfurimonas, Acidovorax, Pelobacter, Sediminibacter, Sinobacteraceae, Desulfobulbus, and Sulfurovum) in the contaminated stations (Fig. 4c). These observations were further supported by clustering and SIMPER analyses. Clustering analysis, based on

Pearson correlation, showed that the lake stations had bacterial communities with different composition to those of the lagoon stations (Fig. 5), which was corroborated by SIMPER analysis (50%). The bacterial communities from the lake stations ST5 and ST8 showed more similar composition (SIMPER: 93%) than those of the lagoon stations ST12 and ST17

(SIMPER: 71%). Consistent with LEfSe, the less contaminated station ST17 exhibited specific dominant OTUs affiliated to Rhodobacter, Planctomycetaceae, Rhizobium, Rhodopirellula, Blastopirellula, Nitrosococcus and Sorangium genera (Fig. 5), which related members are typically detected in marine ecosystems [START_REF] Ben Said | Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia[END_REF][START_REF] Brinkhoff | Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria[END_REF][START_REF] Hernandez-Raquet | Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)[END_REF][START_REF] Jasmin | Diversity of sediment-associated Planctomycetes in the Arabian Sea oxygen minimum zone[END_REF][START_REF] Klotz | Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707[END_REF]. Regarding the contaminated stations, the clustering analysis allowed the identification of specific OTUs for each station. The most PAH-contaminated station ST12 was dominated by specific OTUs belonging to Sediminibacter and Loktanella genera and other genera affiliated to

Rhodobacteraceae, Flavobacteriaceae and Saprospiraceae families (Fig. 5), which related members have been detected in marine sediments after oil spill [START_REF] Acosta-Gonzalez | The effect of oil spills on the bacterial diversity and catabolic function in coastal sediments: a case study on the Prestige oil spill[END_REF][START_REF] Galazka | Genetic and Functional Diversity of Bacterial Microbiome in Soils With Long Term Impacts of Petroleum Hydrocarbons[END_REF][START_REF] Jimenez | The Prestige oil spill: bacterial community dynamics during a field biostimulation assay[END_REF]. The lake stations ST5 and ST8 were dominated by specific OTUs related to Thiobacillus, Nitrospira and Acidovorax genera, Escherichia-Shigella group, and other genera affiliated to Gemmatimonadaceae, Nitrospiraceae, Caldilineaceae and Desulfarculaceae families (Fig. 5), which was consistent with LEfSe (Fig. 4). Related members of these taxa have been found in sediments contaminated by hydrocarbons [START_REF] Jochum | Single-Cell Genomics Reveals a Diverse Metabolic Potential of Uncultivated Desulfatiglans-Related Deltaproteobacteria Widely Distributed in Marine Sediment[END_REF][START_REF] Patel | Taxonomic profiling of bacterial community structure from coastal sediment of Alang-Sosiya shipbreaking yard near Bhavnagar, India[END_REF][START_REF] Reyes-Sosa | Bioprospecting for microbes with potential hydrocarbon remediation activity on the northwest coast of the Yucatan Peninsula, Mexico, using DNA sequencing[END_REF] and pesticides [START_REF] Douglass | Molecular analysis of atrazinedegrading bacteria and catabolic genes in the water column and sediment of a created wetland in an agricultural/urban watershed[END_REF][START_REF] Jiang | Biphenyl-Metabolizing Microbial Community and a Functional Operon Revealed in E-Waste-Contaminated Soil[END_REF].

Although LEfSe revealed that the abundance of some taxa were different between the lake and lagoon ecosystems (Fig. 4), the clustering analysis showed that the four stations shared dominant OTUs affiliated to Acidithiobacilus, Desulfobulbus, Sinobacteraceae, Robiginitalea, Anaerolineaceae and Nannocystineae (Fig. 5). Members of the Acidithiobacillus genera have been detected in metals contaminated ecosystems [START_REF] Giloteaux | Nested PCR and new primers for analysis of sulfate-reducing bacteria in low-cell-biomass environments[END_REF], particularly in sediments [START_REF] Fonti | Influence of biogeochemical interactions on metal bioleaching performance in contaminated marine sediment[END_REF]. It is not surprising to detect metal-resistant bacteria in the Ichkeul Lake / Bizerte Lagoon hydrological system since high levels of metal contents have been detected in the sediments [START_REF] Ben Said | Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon[END_REF][START_REF] Zaaboub | Accumulation of trace metals in sediments in a Mediterranean Lagoon: Usefulness of metal sediment fractionation and elutriate toxicity assessment[END_REF]. The detection of members related to Desulfobulbus, Robiginitalea, Sinobacteraceae, Anaerolineaceae and Nannocystineae encompassing marine species, further support the ecological continuum between the Ichkeul Lake and Bizerte Lagoon. Among them some have been found in contaminated sediments bearing the capacity to degrade hydrocarbons [START_REF] Li | Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing[END_REF][START_REF] Yan | Co-occurrence patterns of the microbial community in polycyclic aromatic hydrocarbon-contaminated riverine sediments[END_REF], and to resist and transform metals [START_REF] Meng | Effects of redox potential on soil cadmium solubility: Insight into microbial community[END_REF][START_REF] Wang | Enhancement of synchronous bio-reductions of vanadium (V) and chromium (VI) by mixed anaerobic culture[END_REF].

Influence of contamination on bacterial assemblages

In order to further decipher the associations between bacterial genera and environmental parameters clustered correlation (ClusCor) analysis was performed at the genus level derived from the OTU abundance table (Fig. 6). specifically abundant for either the lake or the contamination (Fig. 4). Several Pseudomonas and Acidovorax species have been isolated for their ability to degrade pesticides and other organic compounds in diverse habitats [START_REF] Marican | A review on pesticide removal through different processes[END_REF][START_REF] Song | Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments[END_REF].

Interestingly, the presence of the sulfur-oxidizing bacteria related to the Sulfurovum and Sulfurimonas genera suggested the potential anaerobic degradation of pesticides since these genera have been described for their capacity to perform reductive dechlorination of PCB in marine sediments (Matturro et al., 2016b), often associated with sulfate-reducing bacteria (SRB). Also, both genera, Sulfurovum and Sulfurimonas, have been shown to be able to grow under microaerophilic conditions using oxygen or nitrate as electron acceptor [START_REF] Mitchell | The family helicobacteraceae, The Prokaryotes: Deltaproteobacteria and Epsilonproteobacteria[END_REF][START_REF] Xu | Performance and microbial community analysis of a microaerophilic sulfate and nitrate co-reduction system[END_REF]. It is likely that these genera in association with the SRB Desulfarculaceae related genera and Desulfobulbus in the Group1 assemblage might play a role in the sulfur cycle. They probably use the hydrogen sulfur (H2S) produced by SRB and oxygen for their growth playing a role in the degradation of pesticides. The genera related to the Nitrospiraceae have been found dominant in contaminated aquatic ecosystems:

Nitrospira dominant in organic matter contaminated waters [START_REF] Huang | Characterization of sediment bacterial communities in plain lakes with different trophic statuses[END_REF][START_REF] Wang | Response of bacterial community compositions to different sources of pollutants in sediments of a tributary of Taihu Lake, China[END_REF], while Leptospirillum in the presence of metals and metalloids [START_REF] Wang | Bioleaching of Pb-Zn-Sn chalcopyrite concentrate in tank bioreactor and microbial community succession analysis[END_REF].

Also, the presence of Nitrospira together with Thiobacillus, Gemmatimonadaceae, and the genera of the phylum Chloroflexi (Anaerolineaceae and Caldilineaceae), denitrifying genera, suggested a contamination from wastewater treatment systems as previously reported [START_REF] Aanderud | Stoichiometric Shifts in Soil C:N:P Promote Bacterial Taxa Dominance, Maintain Biodiversity, and Deconstruct Community Assemblages[END_REF][START_REF] Zhang | Structure and function of the microbial consortia of activated sludge in typical municipal wastewater treatment plants in winter[END_REF], and further supported by the presence of Escherichia-Shigella and Sinobacteraceae related genera [START_REF] Zhang | Activated sludge bacterial communities of typical wastewater treatment plants: distinct genera identification and metabolic potential differential analysis[END_REF]. Related members of the Nannocystineae genera have been described harbouring the capacity to degrade complex macromolecules [START_REF] Garcia | The family nannocystaceae, The Prokaryotes: Deltaproteobacteria and Epsilonproteobacteria[END_REF]. They have been detected in polluted sediments [START_REF] Acosta-González | Characterization of the anaerobic microbial community in oil-polluted subtidal sediments: aromatic biodegradation potential after the Prestige oil spill[END_REF], but their potential for pollutant degradation still to be demonstrated.

The Group2 included the genera related to Flavobacteriaceae, Flammeovirgaceae, Saprospiraceae, Rhodobacteraceae families, and the genera Actibacter, Sediminibacter, Robiginitalea, Loktanella, Pelobacter, Desulfuromonas. The abundances of Group2 genera showed very strong positive correlation with the concentrations of PAHs and organic-SN compounds (Pearson correlation>0.8), and strong (-0.6>Pearson correlation>-0.8) to very strong (Pearson correlation <-0.8) negative correlations (exclusion) with the concentrations of lindane and other OCPs. These genera, which abundance was correlated to either the lagoon or the contamination level by LEfSe (Fig. 4), have been found in hydrocarbon contaminated marine sediments [START_REF] Acosta-Gonzalez | The effect of oil spills on the bacterial diversity and catabolic function in coastal sediments: a case study on the Prestige oil spill[END_REF][START_REF] Chen See | Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania[END_REF][START_REF] Galazka | Genetic and Functional Diversity of Bacterial Microbiome in Soils With Long Term Impacts of Petroleum Hydrocarbons[END_REF] and oil production facilities [START_REF] Vigneron | Complementary Microorganisms in Highly Corrosive Biofilms from an Offshore Oil Production Facility[END_REF][START_REF] Zapata-Peñasco | Bisulfite reductase and nitrogenase genes retrieved from biocorrosive bacteria in saline produced waters of offshore oil recovery facilities[END_REF]. Particularly interesting, the presence of members related to the Desulfuromonadaceae family (Pelobacter and Desulfuromonas) in this Group2 assemblage that have been described in presence of pesticide and for their capacity of reductive dechlorination [START_REF] Greene | The family desulfuromonadaceae, The Prokaryotes: Deltaproteobacteria and Epsilonproteobacteria[END_REF][START_REF] Sercu | The influence of in situ chemical oxidation on microbial community composition in groundwater contaminated with chlorinated solvents[END_REF].

Such observation suggested that these genera might play a key role to cope with the multicontamination in lagoon sediments.

Behind these two major groups, the ClusCor analyses identified bacterial assemblages (Group3) including genera, which abundances showed mainly strong (-0.6>Pearson correlation>-0.8) to very strong (Pearson correlation<-0.8) negative correlations (exclusion)

with pollutant contents (Fig. 6). The Group3a included genera which abundance showed exclusion with PCBs and PAHs. The abundance of genera from Group3b showed exclusion with the content of all pesticides except heptachlor and strong positive correlation (0.6<Pearson correlation>0.8) with PAHs content (Fig. 6). The Group3c correspond to genera which abundance showed exclusion with PCBs content, while the abundance of genera from Group3d showed exclusion with PCBs and lindane contents (Fig. 6). Noteworthy, most of the genera included in Group3c (Candidatus-Thiobios, Rhizobium, Desulfocapsa, Planctomycetaceae, Rhodopirellula, and Blastopirellula) showed exclusion with pollutants, which was consistent with LEfSe highlighting that the abundance of these genera was correlated with low contamination level (Fig. 4c). It is likely that these genera were sensitive to the presence of pollutants suggesting that their presence might report on ecosystem quality.

However, further investigations are requested to understand their role in the ecosystem functioning.

The co-occurrence analyses showed specific bacterial assemblages linked with contaminants revealing possible metabolic capacities of bacterial genera. Our results are consistent with previous reports that revealed specific populations linked with pollutants in marine ecosystems (Duran et al., 2015a), in freshwater lakes [START_REF] Warnecke | Abundances, identity, and growth state of actinobacteria in mountain lakes of different UV transparency[END_REF], and in soils [START_REF] Llado | Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil[END_REF]. Such information provides new insights useful to guide future research to answer important questions in the field of microbial ecology such as the role of specific populations in the ecosystem functioning and pollutants degradation, and the role of environmental parameters in driving bacterial assemblages.

Conclusions

The determination of bacterial composition in sediment of the Ichkeul Lake / Bizerte Lagoon hydrological system showed that the lake bacterial communities were distinct from those of the lagoon. Although the difference could be attributed to physical-chemical parameters, the salinity and the presence of pollutants playing an important role in structuring the bacterial communities, the study revealed that the lake and the lagoon shared some bacterial genera.

Such result suggested a lake-lagoon continuum due to the hydrology of the system exchanging waters according to the season resulting in bacterial community coalescence. It is likely that such bacterial genera ensure universal biogeochemical functions and, therefore constitute "generalists" bacteria essential for the basic ecosystem functioning. The study also revealed "specialists" bacteria either for the habitat (lake or lagoon) or for the pollutants.

Bacterial "specialists" able to cope with the presence of pesticides in the lake and bacterial "specialists" for PAHs and organic forms of Sn in the Lagoon. These findings represent valuable information, particularly the bacterial "specialists", which represent yet an unexplored metabolic potential for the development of bioremediation processes to mitigate the effect of pollutants in aquatic ecosystems. The study also revealed bacterial genera, which abundance showed exclusion (negative correlation), with the pollutants concentrations. They represent potential bacterial biomarkers reporting on ecosystem quality, which will be useful to develop bacterial tools for monitoring the effect of countermeasures taken for mitigating the pollution. andST8) and Bizerte Lagoon (ST12 and ST17) hydrological system. The analysis was performed at the phylum level applying a threshold similarity of 97 % for OTU identification.

The average of three replicates was used for the analysis. Other, correspond to phyla with relative abundance < 5%. OTUs between stations as well as the specific OTUs for each station. b) Linear discriminant analysis effect size (LEfSe) of the habitat (lake / lagoon). c) LEfSe analysis of the contamination. LEfSe analysis shows the significant differential abundance distribution of bacterial genera between the samples of environmental groups (lake / lagoon; high contamination / low contamination). The analysis was performed applying a threshold similarity of 97 % for OTU identification. The contamination level (high / low) refers to mean effect range medium quotients (m-ERM-Q) presented in Table 1. The analysis was performed applying a threshold similarity of 97 % for OTU identification.

The triplicates for each station are shown in the heatmap. 1. The analysis was performed at the genus level applying a threshold similarity of 97 % for OTU identification. The specific OTUs revealed by LEfSe analysis are coloured according to their specificity: lake (khaki), lagoon (red), high contamination (orange), and low contamination (blue). c Sum of heptachlor and its epoxide derivative.

d Sum of lindane and its metabolites.

e Sum of endosulfan, dieldrin, aldrin, chlordane, and their derivatives. g m-ERM-Q, mean effect range medium quotient calculated according to [START_REF] Long | Predicting toxicity in marine sediments with numerical sediment quality guidelines[END_REF].

h Quality classification according to [START_REF] Bakke | Development of sediment quality criteria in Norway[END_REF].

  First, sequences related to 11 genera including Sulfurovum, Pseudomonas, Leptospirillum, Sulfurimonas, Acidovorax, and Nitrospira genera, and genera related to Anaerolineaceae, Desulfarculaceae, Caldilineaceae, Gemmatimonadaceae, and Nitrospiraceae compose the Group1a. The abundance of the Group1a genera showed strong (0.6>Pearson correlation<0.8) to very strong (Pearson correlation>0.8) positive correlations with the concentration of the different pesticides (PCBs, Lindane, and other OCPs), and strong (-0.6>Pearson correlation>-0.8) to very strong (Pearson correlation<-0.8) negative correlation (exclusion) with PAHs concentrations. This Group1a was closely related with the Group1b including Desulfobulbus and Thiobacillus genera, and genera related to Nannocystineae, Sinobacteraceae, and Escherichia-Shigella, which abundances showed strong (0.6>Pearson correlation<0.8) to very strong (Pearson correlation>0.8) positive correlations with the concentrations of pesticide compounds and strong negative correlation (-0.6>Pearson correlation>-0.8, i.e. exclusion) with the other pollutants. Consistently, LEfSe identified all these genera (Group1a and Group1b)

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Comparison of bacterial communities from stations of the Ichkeul Lake / Bizerte Lagoon hydrological system. The canonical correspondence analysis shows the correlations between bacterial communities structures (T-RFLP patterns) and environmental chemical data in a) the Bizerte Lagoon-Ichkeul Lake complex, b) in Ichkeul Lake sediments,and c) in Bizerte Lagoon sediments. The analyses were performed in September 2011 (wet season) and April 2012 (dry season). Lake September, pink circles; Lake April, blue circles; Lagoon September, red diamonds; Lagoon April brown diamonds. The analyses are based on biological triplicates for each station. ∑ Pesticides, total pesticides; ∑ PAHs, total PAHs; ∑ PCBs, total PCB; ∑ Sn, total Sn organic compounds; T°, temperature; m-ERM-Q, mean effect range medium quotient; P, phosphate; DO, dissolved oxygen. The names of the stations further characterized in this study are indicated ST5 and ST8 for the Ichkeul Lake, and ST12 and ST17 for the Bizerte Lagoon.

Figure 3 .

 3 Figure 3. Composition of bacterial communities from stations of the Ichkeul Lake (ST5

Figure 4 .

 4 Figure 4. Comparison of bacterial communities from the different stations of the Ichkeul Lake / Bizerte Lagoon hydrological system. a) Venn diagram showing the number of shared

Figure 5 .

 5 Figure 5. Heatmap comparing the bacterial communities from the different stations of

Figure 6 .

 6 Figure 6. Clustered correlation (ClusCor) between bacterial OTUs and pollutants

f

  Sum of PAHs including naphthalene, acenaphtylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pryrene, indenol[1,2,3-cd]pyrene, dibenz[a,h]anthracene, and benzoghiperylene.

  Table 1 summarizes the chemical data showing the sum of the different

	types of pollutants obtained in April 2012 for four representative and contrasted stations: 2
	from the lake (ST5 and ST8) and 2 from the lagoon (ST12 and ST17). The Bizerte Lagoon
	was characterized by PAH concentrations ranging from 137 ± 26 to 17988 ± 1183 ng g -1 ,
	corresponding to class I (ST12, insignificantly polluted) and class IV (ST17, severely

polluted), respectively according to the classification developed by

[START_REF] Bakke | Development of sediment quality criteria in Norway[END_REF]

. Both stations of the Ichkeul Lake (ST5 and ST8) were classified as class II with PAH concentrations of 1362 ± 560 and 444 ± 24 ng g -1 , respectively. Considering the contamination by organochlorine pesticides (OCPs), the Ichkeul Lake stations were classified as class IV (severely polluted) while the Bizerte Lagoon stations were classified as class III (markedly polluted) according to the

[START_REF] Bakke | Development of sediment quality criteria in Norway[END_REF]

classification. The OCPs, including

Table 1 : Chemical analyses of contaminants in the Ichkeul Lake / Bizerte Lagoon hydrological system and sediment toxicity potential

 1 . The data corresponds to the sum of the different types of pollutants obtained in April 2012 described in details by Ben[START_REF] Salem | Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia[END_REF]. Average ± SD are presented (n=3).

	Pollutants (ng g -1 dry wt)	ST5	Lake	ST8	Stations	ST12	Lagoon	ST17
	Organic Sn a	143±0.3			83±0.3	154±0.1	102±0.5
	PCBs b	4.66±0.7			5.58±1	4.54±0.1	2.48±0.8
		(I) h			(II) h			(I) h	(I) h
	Heptachlor	10.7±0.7			14±1.6		11±4	12±4
	Lindane d	268±6			283±13	101±5	95±14
	Other OCPs e	586±0.03		1717±65	53±3.4	86±7.4
		(IV) h			(IV) h			(III) h	(III) h
	PAHs f	1362±560		444±24	17988±1183	137±26
		(II) h			(II) h			(IV) h	(I) h
	m-ERM-Q g	8.73±0.03	13.30±0.4	5.30±0.06	3.90±0.1
	Sum of PCB congeners including PCB18, PCB28, PCB31, PCB44, PCB52, PCB101, PCB138, PCB118,
	PCB153, PCB149, PCB180, PCB194.

a sum of organic forms of tin including methyl, butyl, and phenyl derivatives. b

Table 2 : Alpha diversity indices (mean ± SD, n=3) of microbial community from the Ichkeul Lake / Bizerte Lagoon stations

 2

			Station
	Indices	ST5	ST8	ST12	ST17
			Lake	Lagoon
	Reads a	22368	19247	22101	25662
	Trimmed sequences b 16378	14217	15185	18502
	Species richness (R) 347 ± 32	359 ± 28	341 ± 27	356 ± 40
	Coverage	0.93 ± 0.01 0.93 ± 0.01 0.94 ± 0.01 0.94 ± 0.01
	Simpson (1-D)	0.97 ± 0.01 0.98 ± 0.01 0.96 ± 0.01 0.98 ± 0.01
	Shannon (H)	4.51 ± 0.1	4.58 ± 0.1	4.37 ± 0.1	4.51 ± 0.05
	Evenness (E)	0.26 ± 0.04 0.27 ± 0.01 0.23 ± 0.03 0.26 ± 0.02
	Chao-1	373 ± 39	386 ± 27	362 ± 27	379 ± 45

a SD: ST5, ± 1966; ST8, ± 1866; ST12, ± 3380; ST17, ± 1688. b SD: ST5, ± 1481; ST8, ± 1406; ST12, ±2708; ST17, ± 1611.

Acknowledgements

The authors would like to thank Mme Nabiha M'barek (National Agency of Environment Protection Tunisia) and Mr. Taher Kaabi (Commissariat Regional Commission for Agricultural Development of Bizerte) for their logistic contribution. We acknowledge the Regional Platform for Environmental Microbiology PREMICE supported by the Aquitaine Regional Government Council and the urban community of Pau-Pyrénées.

Funding information

This study was funded by the European programme ERANET-MED through the AQUASALT (NMED-0003-01) project.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.