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The ability of WWTP outflow bacteria at colonizing rock surfaces and contributing to the formation of river epilithic biofilms was investigated. Bacterial community structures of biofilms (b-) developing on rocks exposed to treated wastewaters (TWW) of a hospital (HTWW) and a domestic (DTWW) clarifier, and to surface waters of the stream located at 10 m, 500 m, and 8 km from the WWTP outlet, were compared. Biofilm bacterial contents were analyzed by cultural approaches and a tpm-based DNA metabarcoding analytical scheme. Co-occurrence distribution pattern analyses between bacterial datasets and eighteen monitored pharmaceuticals were performed. Higher concentrations of iohexol, ranitidine, levofloxacin, and roxithromycin were observed in the b-HTWW while atenolol, diclofenac, propranolol, and trimethoprim were higher in the b-DTWW. MPN growth assays showed recurrent occurrences of Pseudomonas aeruginosa and Aeromonas caviae among these biofilms. An enrichment of multi-resistant P. aeruginosa cells was observed in the hospital sewer line. P. aeruginosa MPN values were negatively correlated to roxithromycin concentrations. The tpm DNA metabarcoding analyses confirmed these trends and allowed an additional tracking of more than 90 species from 24 genera. Among the recorded 3082 tpm ASV (amplicon sequence variants), 41% were allocated to the Pseudomonas. Significant differences through ANOSIM and DESeq2 statistical tests were observed between ASV recovered from b-HTWW, b-DTWW, and epilithic river biofilms. More than 500 ASV were found restricted to a single sewer line such as those allocated to Aeromonas popoffii and Stenotrophomonas humi being strictly found in the b-HTWW file. Several significant correlations between tpm ASV counts per species and pharmaceutical concentrations in biofilms were recorded such as those of Lamprocystis purpurea being positively correlated with trimethoprim concentrations. A tpm source tracking analysis showed the b-DTWW and b-HTWW tpm ASV to have contributed, respectively, at up to 35% and 2.5% of the epilithic river biofilm tpm-taxa recovered downstream from the WWTP outlet. Higher contributions of TWW taxa among epilithic biofilms were recorded closer to the WWTP outlet. These analyses demonstrated a coalescence of WWTP sewer communities with river freshwater taxa among epilithic biofilms developing downstream of a WWTP outlet.

International audience; Active functional groups in biofilms determine the adsorption and desorption of contaminants and nutrients. Epilithic biofilms were characterized in order to understand the association between the chemistry alteration patterns and the surrounding anthropic activities of the Guaporé River watershed. The instrumental analyses included pyrolysis coupled to gas chromatography and mass spectroscopy, spectroscopy in the IR region with attenuated total reflectance and two-dimensional nuclear magnetic resonance. Spectrometric techniques demonstrated that epilithic biofilms are mainly composed of polysaccharides, nitrogencontaining compounds, lipids, and aromatic and phenolic structures, which have functional groups characteristic of alcohols, esters, ethers and amides. The polysaccharide levels reflect well the environmental pressures. The chemical composition of epilithic biofilms can be an effective tool for environmental assessment in watercourses, since the different anthropic actions developed in watersheds, mainly agriculture and urban areas, can modify the organic fraction of biofilms.

International audience; In forested headwater streams, inconspicuous food resources such as epilithic microalgae can play a major role owing to their content of long chain polyunsaturated fatty acids (PUFAs) that are essential for macroinvertebrate development. Yet, the use of these resources and their consequences for consumers and life-history traits remain scarcely studied, especially for non-herbivorous taxa. Using instream mesocosms, we aimed to understand how two macroinvertebrate species, a shredder detritivore (Gammarus pulex) and a scraper (Rhithrogena semicolorata), use available organic resources under light versus shaded conditions (high and low amounts of phototrophic biofilm, respectively). We specifically focused on the origin of carbon (C) and essential lipids (PUFAs, sterols) assimilated by the two species and the effects of these compounds on their survival and growth. When autotrophic biofilms were available (mesocosms exposed to light), both species experienced significantly higher growth than in mesocosms placed in the dark. In Rhithrogena nymphs, the survival and imago emergence rates were positively affected by access to autotrophic biofilms. Using stable isotope analysis (δ13C and δ15N), we demonstrated that under dark conditions both macroinvertebrates assimilated C of detrital origin. Under light conditions, most of the C assimilated by R. semicolorata was derived from autotrophic biofilms and around 11% from fine detrital particles (FPOM). Gammarus pulex derived part of its C from detrital sources (mainly FPOM) and from autotrophic biofilms. The results of isotopic analyses (δ13C) on fatty acids (FAs) and sterols showed that the scraper Rhithrogena was entirely dependent on autotrophic biofilms to meet its dietary FAs and sterol requirements. In contrast, detrital sources were quantitatively important for G. pulex both in terms of C and sterol supply, irrespective of the conditions tested. For these consumers, microalgae seemed to be a complementary food source, but yet essential to cover a large part of their requirements in long-chain PUFAs. This study clearly confirmed the ecological importance of autotrophic biofilms for two functional feeding groups of macroinvertebrates in headwater streams providing new insights on the trophic origin of sterols and long-chain PUFAs in their diet.

In headwater streams, macroinvertebrates play an essential role in the flow of matter and energy from basal organic sources to the upper trophic level (fish fauna). Macroinvertebrate’s development depends on a limited number of basal sources from allochthonous leaf litter to autochthonous primary production (benthic microalgae and bryophytes). The riparian forest and tree cover of these small streams produce a substantial input of allochthonous detrital carbon, which is regarded as the basis for ecosystem functioning according to the RCC (river continuum concept). However, the concept outlined by the RPM (riverine productivity model) and recent studies tend to emphasise the importance of autochthonous primary production and particularly that of diatoms, which appear to be able to sustain secondary production thanks to its nutritional quality that is clearly higher than that of detrital material. In this context, the aim of this phd-thesis was to study the trophic relationships between the main basal sources and a selection of macroinvertebrates typical of headwater streams. The focus was on the importance of primary producers and benthic microalgae, in the trophic functioning of these environments, in particular by providing essential molecules (polyunsaturated fatty acids and sterols). First, in a mesocosm experiment, we studied the use of available organic resources by the scraper, Rhithrogena semicolorata and the shredder, Gammarus pulex, in the presence or absence of autotrophic biofilms. By using an approach based on stable isotopes analysis (��13C & ��15N) of bulks, fatty acids and sterols, we demonstrated that microalgae (diatoms) were assimilated by both species and their ingestion was essential to Rhithrogena semicolorata in relation to the supply of essential fatty acids (20:5ω3 and 20:4ω6). For Gammarus pulex, the feeding on microalgae was not essential when leaf litter was not recalcitrant but promoted a higher somatic growth. In a second time, a seasonal monitoring was undertaken on a mid-mountain forested stream (Massif Central, France). In early spring, using a Bayesian mixture model (SIAR), we observed that most of the energy assimilated by nine macroinvertebrate taxa (of different trophic guilds) originated from epilithic biofilms, bryophytes and fine benthic detritus particles. Using stable carbon isotope signatures of fatty acids (CSIA, ��C13FA), we showed that current fatty acids (16:0, 18:1ω9, 18:2ω6 and 18:3ω3) were supplied in proportion to the different organic sources assimilated by invertebrates. In contrast, the essential fatty acids (20:4ω6 and 20:5ω3) were derived from autochthonous sources and obtained through different feeding strategies, such as omnivory. The follow-up during summer and autumn, but on a smaller number of macroinvertebrate taxa (five), confirmed the observations from the spring. Epilithic and epixylic biofilms were still the source of essential fatty acids and notably 20:4ω6 and 20:5ω3 assimilated by the different macroinvertebrate taxa, irrespective of their usual trophic guild. Furthermore, we demonstrated that although bryophytes and fine detrital particles were significant food sources, the recalcitrant leaf litter from the adjacent beech forest was little assimilated by macroinvertebrate shredders. Lastly, the analysis of sterol transfer, both under natural conditions and during the mesocosm experiment, suggested that no nutritional constraints linked to these compounds occurred among all the macroinvertebrate species analysed.; Dans les cours d’eau de tête de bassin versant, les macroinvertébrés exercent une fonction essentielle dans les flux de matières et d’énergie allant des sources organiques basales au maillon trophique supérieur que constitue la faune piscicole. Leur développement dépend d’un nombre restreint de sources organiques d’origine allochtone (litière de feuilles) ou autochtone (microalgues benthiques et bryophytes). L’environnement forestier et les ripisylves arborées de ces petits cours d’eau engendrent un apport prépondérant de carbone détritique allochtone habituellement considéré comme étant la base du fonctionnement de l’écosystème selon le RCC (river continuum concept). Cependant, le concept décrit par le RPM (riverine productivity model) et des études récentes mettent en avant l’importance de la production primaire autochtone et en particulier celle des diatomées, qui paraissent apte à soutenir la production secondaire grâce à une qualité nutritionnelle nettement supérieure à celle du matériel détritique. Dans ce contexte, ce travail de thèse avait pour objectif d’étudier les relations trophiques qui s’établissent entre les principales sources basales et une sélection de macroinvertébrés inféodés aux cours d’eau de tête de bassin versant. L’accent a été mis sur l’importance que pouvait représenter les producteurs primaires et plus particulièrement les microalgues benthiques dans le fonctionnement trophique de ces milieux, en assurant notamment la couverture en molécules essentielles (acides gras polyinsaturés et stérols). Dans un premier temps, au cours d’une expérimentation menée en mésocosmes, nous avons étudié chez le racleur, Rhithrogena semicolorata et le broyeur, Gammarus pulex, l’utilisation des ressources organiques disponibles en présence ou pas de biofilms autotrophes. Grâce à une approche reposant sur les isotopes stables (��13C & ��15N) des organismes, des acides gras et des stérols, nous avons pu démontrer que les microalgues (diatomées) étaient assimilées par les deux espèces et leur consommation avait un caractère essentiel chez Rhithrogena semicolorata en lien avec l’apport en acides gras essentiels (20:5ω3 et 20:4ω6). Dans le cas de Gammarus pulex, la consommation de microalgues était non-essentielle en présence de litière de feuilles peu récalcitrantes, mais favorisait la croissance somatique. Dans un second temps, un suivi saisonnier a été entrepris sur un ruisseau forestier de moyenne montagne (Massif Central). Au début du printemps, grâce à un modèle de mélange bayésien (SIAR) nous avons mis en évidence que l’essentiel de l'énergie reçu par neuf taxons de macroinvertébrés (de guilde trophique différente) provenait majoritairement des biofilms épilithiques, des bryophytes et de fines particules détritiques benthiques. L’utilisation des signatures isotopique du carbone des acides gras (AICS, ��C13AG), a permis de montrer que les acides gras courants (16:0, 18:1ω9, 18:2ω6 et 18:3ω3) étaient amenés à proportion des différentes sources organiques assimilées par les invertébrés. En revanche, les acides gras essentiels (20:4ω6 et 20:5ω3) provenaient des sources autochtones, obtenus par l’intermédiaire de différentes stratégies alimentaire, tel que l’omnivorie. La poursuite de ce suivi en été et en automne, mais sur un nombre plus réduit de macroinvertébrés (cinq) est venue confirmer les observations faites au printemps. Les biofilms épilithiques et épixyliques étaient toujours à l’origine des acides gras essentiels et notamment du 20:4ω6 et du 20:5ω3 assimilés par les différents taxons de macroinvertébrés, indépendamment de leur guilde trophique. De plus, il a pu être démontré que si les bryophytes et les fines particules détritiques constituaient une source d’alimentation non négligeable, les litières de feuilles récalcitrantes issues de la hêtraie environnante étaient peu assimilées par les macroinvertébrés broyeurs. (...)

International audience; The rivers of Guadeloupe and Martinique (French West Indies) show high levels of chlordecone (CLD) contamination. This persistent molecule has a dramatic impact on both aquatic ecosystems and human health. In these rivers, epilithic biofilms are the main endogenous primary producers and represent a central food source for fish and crustaceans. Recently, their viscoelastic properties have been shown to be effective in bio-assessing pollution in tropical environments. As these properties are closely related to the biochemical composition of the biofilms, biochemical (fatty acids, pigments, extracellular polymeric substances (EPS) monosaccharides) and molecular markers (T-RFLP fingerprints of bacteria, archaea and eukaryotes) were investigated. Strong links between CLD pollution and both biofilm biochemistry and microbial community composition were found. In particular, high levels of CLD were linked with modified exo-polysaccharides corresponding to carbohydrates with enhanced adsorption and adhesion properties. The observed change probably resulted from a preferential interaction between CLD and sugars and/or a differential microbial secretion of EPS in response to the pollutant. These changes were expected to impact viscoelastic properties of epilithic biofilms highlighting the effect of CLD pollution on biofilm EPS matrix. They also suggested that microorganisms implement a CLD scavenging strategy, providing new insights on the role of EPS in the adaptation of microorganisms to CLD-polluted environments.

Epilithic biofilms are communities of microorganisms composed mainly of microbial cells, extracellular polymeric substances from the metabolism of microorganisms, and inorganic materials. Biofilms are a useful tool to assess the impact of anthropic action on aquatic environments including the presence of pesticide residues such as glyphosate. The present work seeks to monitor the occurrence of glyphosate and AMPA residues in epilithic biofilms occurring in a watershed. For this, epilithic biofilm samples were collected in the Guaporé River watershed in the fall and spring seasons of 2016 at eight points. Physicochemical properties of the water and biofilms were determined. The determination of glyphosate and AMPA was performed using an ultra-high performance liquid chromatograph coupled to a tandem mass spectrometer. The concentrations of glyphosate and AMPA detected in epilithic biofilms vary with the season (from 90 to 305 μg kg−1 for glyphosate and from 50 to 240 μg kg−1 for AMPA, in fall and spring, respectively) and are strongly influenced by the amount of herbicide applications. Protected locations and those with poor access not demonstrate the presence of these contaminants. In the other seven points of the Guaporé River watershed, glyphosate was detected in concentrations ranging from 10 to 305 μg kg−1, and concentrations of AMPA ranged from 50 to 670 μg kg−1. An overview of the contamination in the Guaporé watershed shows that the most affected areas are located in the Marau sub-watershed, which are strongly influenced by the presence of the city of Marau. This confirms the indiscriminate use of glyphosate in the urban area (weed control, domestic gardens and horticulture) and constitutes a problem for human and animal health. The results showed that biofilms can accumulate glyphosate resulting from the contamination of water courses and are sensitive to the sources of pollution and pesticides present in rivers. Fil: Fernandes, Gracieli. Universidade Federal de Santa Maria; Brasil Fil: Aparicio, Virginia Carolina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Agronomía; Argentina Fil: Bastos, Marilia Camotti. Université de Lorraine; Francia Fil: de Gerónimo, Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina Fil: Labanowski, Jérôme. Université de Poitiers; Francia Fil: Prestes, Osmar Damian. Universidade Federal de Santa Maria; Brasil Fil: Zanella, Renato. Universidade Federal de Santa Maria; Brasil Fil: dos Santos, Danilo Rheinheimer. Universidade Federal de Santa Maria; Brasil

A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidetes . To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus ) was 34% and NRPS genes were found in 78%. In Proteobacteria , PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria , respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes. A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus) was 34% and NRPS genes were found in 78%. In Proteobacteria, PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria, respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.

Soil and rock surfaces support microbial communities involved in mineral weathering processes. Using selective isolation, fungi were obtained from limestone surfaces of Mayan monuments in the semi-arid climate at Yucatan, Mexico. A total of 101 isolates representing 53 different taxa were studied. Common fungi such as Fusarium, Pestalotiopsis, Trichoderma, and Penicillium were associated with surfaces and were, probably derived from airborne spores. In contrast, unusual fungi such as Rosellinia, Annulohypoxylon, and Xylaria were predominantly identified from mycelium particles of biofilm biomass. Simulating oligotrophic conditions, agar amended with CaCO3 was inoculated with fungi to test for carbonate activity. A substantial proportion of fungi, in particular those isolated from mycelium (59%), were capable of solubilizing calcium by means of organic acid release, notably oxalic acid as evidenced by ion chromatography. Contrary to our hypothesis, nutrient level was not a variable influencing the CaCO3 solubilization ability among isolates. Particularly active fungi (Annulohypoxylon stygium, Penicillium oxalicum, and Rosellinia sp.) were selected as models for bioweathering experiments with limestone-containing mesocosms to identify if other mineral phases, in addition to oxalates, were linked to bioweathering processes. Fungal biofilms were seen heavily covering the stone surface, while a biomineralized front was also observed at the stone-biofilm interface, where network of hyphae and mycogenic crystals was observed. X-ray diffraction analysis (XRD) identified calcite as the main phase, along with whewellite and wedellite. In addition, lower levels of citrate were detected by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Overall, our results suggest that a diverse fungal community is associated with limestone surfaces insemi-arid climates. A subset of this community is geochemically active, excreting organic acids under quasi-oligotrophic conditions, suggesting that the high metabolic cost of exuding organic acids beneficial under nutrient limitation. Oxalic acid release may deteriorate or stabilize limestone surfaces, depending on microclimatic dynamics.

Epilithic bacterial community viability was assessed on natural biofilm assemblages from environmentally contrasting locations over a 17-months period to determine if it reflects environmental conditions or conditions within the biofilm assemblage. Vital state was assessed by membrane integrity using LIVE/DEAD® BacLight™ staining kit. Samples were regularly collected in a large river, up and downstream of a large urban centre. Epilithic biomasses were similar between sites irrespective of the distinct water quality but varied temporarily, peaking up to 48 g AFDM m−2. Bacterial community composition assessed by 16S rDNA based PCR-DGGE significantly differed between sites. Bacterial densities (median of 2.5 × 1011 cell g AFDM−1) were stable whatever the sample origin or biomass. Viable bacterial fractions ranged between 13 and 83% of the total bacterial densities and were correlated with hydrological stability indicators (average of 41.9% during stable water periods, 62.4% during intermediate flow regimes and 50.0% during flow instability) and seasonal parameters. At the river section and epilithic community scales, consistent bacterial densities per unit of biomass could reflect a biofilm assemblage carrying capacity while variable membrane integrity likely integrates changes in the vital state of the community under changing environmental conditions.

International audience; Phototrophic river biofilms are microbial assemblages involved in in-stream processes. For a better understanding of N-cycling, the denitrifier community abundance, activity and structure were monitored in natural biofilm assemblages, in two sites exhibiting contrasting nutrient concentrations during a 1-year period. The denitrifier abundance, estimated by quantification of the nirS, nirK and nosZ genes, correlated to biofilm biomass and total bacterial counts. Site-related differences in denitrification activity were observed and the rates were significantly correlated with the nosZ gene copy numbers and biofilm biomass. The denitrifier community structure, assessed by PCR-DGGE of nosZ, differed between sites with only minor differences between sampling occasions, and correlated with the total bacterial community structure. Altogether, these findings suggest that nutrient loading, especially nitrogen, affect both denitrifier community structure and activity.

International audience; The present study sought to identify a minimal adequate model to describe the biomass dynamics of river epilithon, a functional indicator of river health. Identification of minimal adequate models is particularly necessary in river management, given the reduced number of variables authorities are willing to measure routinely. A model previously developed for epilithon dynamics in a pre-alpine river was applied to epilithon biomasses recorded in contrasting hydrological, trophic and light conditions at various sites in the Agu¨era stream (Spain) over 3 years (11 case studies). A model selection tool, the Akaike Information Criterion (AIC), was used to determine the optimal combination of parameters. In nine of 11 case studies, the best model described epilithon biomass dynamics as the equilibrium between phototrophic growth and discharge-dependent loss and ignored light, temperature and nutrient influences. The best adequate minimal model i.e. the model that is the best trade-off between goodness-of-fit and model simplicity performed best, in years in which clearly contrasting short low and high water periods occurred. During years with less marked hydrodynamics, many other abiotic or biotic processes influenced epilithon biomass dynamics. In these cases, weaker goodness-of-fit had to be accepted to avoid excessively increasing model complexity.

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.