Determinism of bacterial dynamics in microphytobenthic biofilms from intertidal mudflats : role of extracellular polymeric substances

Les vasières intertidales sont le siège d’une forte production primaire sous la forme d’un biofilmmicrophytobenthique qui se développe en surface à marée basse. Ce biofilm se compose principalement de diatomées et de bactéries hétérotrophes. Ces deux composantes sécrètent des substances polymériques extracellulaires (EPS) qui jouent différents rôles dans le biofilm. Le travail présenté s’est basé sur différentes échelles d’observation (in situ à 2 saisons, mésocosme en laboratoire, et échelle fine du biofilm en microscopie confocale) et a permis de mettre en évidence les interactions des diatomées et des bactéries à différents moments de la marée et du nycthémère. L’utilisation d’une nouvelle méthode d’extraction des EPS a permis d’éclaircir leur rôle en évitant les contaminations par les substances internes provoquées par les méthodes classiques. Les EPS colloïdales particulièrement riches en glucose s’associent au déplacement des diatomées, notamment lors d’un stress (sursalure, carence en nutriments) et sont préférentiellement consommées par les bactéries, après leur dégradation par les enzymes ou leur hydrolyse dans l’eau interstitielle. Les EPS liées au frustule, et plus particulièrement les sucres, inhiberaient le développement bactérien à proximité de la cellule algale. Elles sont surtout sécrétées lors de la mise en place du biofilm et pour protéger la cellule quand les conditions sont osmotiquement défavorables et leur richesse en protéine leur confère un potentiel intéressant pour les bactéries qui peuvent les utiliser comme substrat azoté en cas de carence. D’autres substances ont été plutôt sécrétées parles bactéries telles que les N-acétylglucosamines et les protéines colloïdales de bas poids moléculaire,certainement des enzymes bactériennes, ainsi que le glucose qui semble être associé au EPS colloïdaux des diatomées mais aussi aux EPS bactériens selon l’analyse en microscopie confocale en utilisant des lectines comme marqueurs d’EPS.Microphytobenthic biofilms that develop at the intertidal mudflat surface are responsible of a substantialprimary production. These biofilms are composed of diatoms and heterotrophic bacteria, both excretingextracellular polymeric substances (EPS) that have various functions into the biofilm. The present thesis is basedon different observation scales that have highlighted the complex interactions between diatoms and bacteria fordifferent moment of the biofilm development (in situ at 2 seasons, in laboratory mesocosm and at the scale of thebiofilm at confocal microscopy). Furthermore, a new extraction method that avoids contamination with internalsubstances was used and provides new insights about the role of EPS. Colloidal EPS are excreted during thediatom migrations, notably in case of environmental stress (nutrient depletion, high salinity). They arepreferentially consumed by bacteria, after hydrolysis or enzymatic cleavage. Bound EPS are associated to abacterial inhibition, especially sugars. These EPS are mainly excreted during biofilm formation and also in caseof osmotic stress, if diatoms cannot migrate toward favorable slices. Some substances are associated to bacterialdevelopment, such as N-acetylglucosamine, low molecular weight proteins that probably consist of bacterialenzymes and also glucose that seems to be associated to colloidal EPS secreted by diatoms as well as bacterialEPS, when analyzing the biofilm with confocal microscopy by using lectins as EPS biomarkers

Déterminisme de la production bactérienne dans les vasières intertidales du Bassin de Marennes-Oléron : rôle des exopolysaccharides

Microphytobenthic biofilms that develop at the intertidal mudflat surface are responsible of a substantialprimary production. These biofilms are composed of diatoms and heterotrophic bacteria, both excretingextracellular polymeric substances (EPS) that have various functions into the biofilm. The present thesis is basedon different observation scales that have highlighted the complex interactions between diatoms and bacteria fordifferent moment of the biofilm development (in situ at 2 seasons, in laboratory mesocosm and at the scale of thebiofilm at confocal microscopy). Furthermore, a new extraction method that avoids contamination with internalsubstances was used and provides new insights about the role of EPS. Colloidal EPS are excreted during thediatom migrations, notably in case of environmental stress (nutrient depletion, high salinity). They arepreferentially consumed by bacteria, after hydrolysis or enzymatic cleavage. Bound EPS are associated to abacterial inhibition, especially sugars. These EPS are mainly excreted during biofilm formation and also in caseof osmotic stress, if diatoms cannot migrate toward favorable slices. Some substances are associated to bacterialdevelopment, such as N-acetylglucosamine, low molecular weight proteins that probably consist of bacterialenzymes and also glucose that seems to be associated to colloidal EPS secreted by diatoms as well as bacterialEPS, when analyzing the biofilm with confocal microscopy by using lectins as EPS biomarkers.Les vasières intertidales sont le siège d’une forte production primaire sous la forme d’un biofilmmicrophytobenthique qui se développe en surface à marée basse. Ce biofilm se compose principalement de diatomées et de bactéries hétérotrophes. Ces deux composantes sécrètent des substances polymériques extracellulaires (EPS) qui jouent différents rôles dans le biofilm. Le travail présenté s’est basé sur différentes échelles d’observation (in situ à 2 saisons, mésocosme en laboratoire, et échelle fine du biofilm en microscopie confocale) et a permis de mettre en évidence les interactions des diatomées et des bactéries à différents moments de la marée et du nycthémère. L’utilisation d’une nouvelle méthode d’extraction des EPS a permis d’éclaircir leur rôle en évitant les contaminations par les substances internes provoquées par les méthodes classiques. Les EPS colloïdales particulièrement riches en glucose s’associent au déplacement des diatomées, notamment lors d’un stress (sursalure, carence en nutriments) et sont préférentiellement consommées par les bactéries, après leur dégradation par les enzymes ou leur hydrolyse dans l’eau interstitielle. Les EPS liées au frustule, et plus particulièrement les sucres, inhiberaient le développement bactérien à proximité de la cellule algale. Elles sont surtout sécrétées lors de la mise en place du biofilm et pour protéger la cellule quand les conditions sont osmotiquement défavorables et leur richesse en protéine leur confère un potentiel intéressant pour les bactéries qui peuvent les utiliser comme substrat azoté en cas de carence. D’autres substances ont été plutôt sécrétées parles bactéries telles que les N-acétylglucosamines et les protéines colloïdales de bas poids moléculaire,certainement des enzymes bactériennes, ainsi que le glucose qui semble être associé au EPS colloïdaux des diatomées mais aussi aux EPS bactériens selon l’analyse en microscopie confocale en utilisant des lectines comme marqueurs d’EPS

Viral impact on prokaryotic and microalgal activities in the microphytobenthic biofilm of an intertidal mudflat (French Atlantic coast)

International audienceThis is the first report on viriobenthos activity within the microbial biofilm located at the top-surface of the intertidal mudflat during emersion in Marennes-Oléron Bay (France). By combining in situ and ex situ approaches, the viral production (VP) was linked to the dynamics of prokaryotes and microphytobenthos (MPB). VP averaged 2-4 x 108 viruses ml-1 h-1. VP correlated positively with the Virus to Prokaryote Ratio, and both were correlated negatively with the water content. The virus-induced mortality of prokaryotes was lower in winter than in summer (6.8 vs 39.7 % of the production) and the C-shunting may supply 2 to 12% of their Carbon Demand, respectively. VP accounted for 79% of loss in Prokaryotes but the response was delayed compared to the increase in VP suggesting a simultaneous release of viruses of MPB origin. This hypothesis is supported by capsid-sizing of virions by transmission electronic microscopy and bioassays. Harvesting and ex situ maintenance of top-surface sediments was carried out to monitor the dynamics of viruses, prokaryotes and MPB after inoculation with benthic or planktonic viruses. Benthic viruses modified the prokaryotic and MPB dynamics and decreased the photosynthesis efficiency in contrast to planktonic viruses that impacted MPB but not the prokaryotes

A method to reconstruct anguilliform Wshes from partially digested items

International audienceEstimating the amounts of resources consumed by individuals is important in many studies. For predators, allometric relationships can be used to extrapolate the size of preys from undigested remains found in the stomach and in the faeces. However, such equations are available for a limited number of species. Based on a large sample size gathered in New Caledonia on both predators (sea kraits) and their preys (anguilliform Wshes), we provide the Wrst allometric relationships that allow estimating accurately the mass and the size of various anguilliform Wsh species

Laticauda saintgironsi (sea krait). Predation.

National audienc

Bacterial Dynamics in a Microphytobenthic Biofilm: a Tidal Mesocosm Approach

In intertidal mudflats, during low tide exposure, microphytobenthos (MPB) migrate vertically through the surface sediment and form, with the heterotrophic bacteria, a transient biofilm. Inside this biofilm, multiple interactions exist between MPB and bacteria. These micro-organisms secrete a wide range of extracellular polymeric substances (EPS), which are major components of the biofilm matrix. In this study, we used a tidal mesocosm experiment in order to decipher the interactions of the MPB-EPS-bacteria complex within the biofilm. We tried to determine if the EPS could control bacterial activities and/or production and/or richness according to the age of the biofilm and to the immersion/emersion period. The dynamics of biomasses of MPB and prokaryotes, the bacterial production, the hydrolysis of predominating organic constituents in the dissolved organic carbon (DOC) pool (i.e., carbohydrates and polypeptides), and the bacterial structure were studied in relation to the different EPS fractions (carbohydrates and proteins: colloidal and bound) dynamics during 8 days. Our experiment had emphasized the influence of the environmental conditions (light, immersion/emersion) on the interactions within the biofilm and also on the effects on biofilm ageing. Bacterial production was always inhibited by the bound EPS-carbohydrate, especially during low tide. Our results suggest that the concentration and composition of EPS had a major role in the bacterial/MPB interactions: these interactions can be either positive or negative in order to regulate the productive phases of MPB and bacteria

Tidal and seasonal effects on the short-term temporal patterns of bacteria, microphytobenthos and exopolymers in natural intertidal biofilms (Brouage, France)

International audienceRelationships between bacteria, microphytobenthos and extracellular polymeric substances (EPS) that makeup microbial biofilms over bare mudflats were investigated at an hourly frequency during two 14-day spring–neap cycles in winter and summer 2008. Bacterial abundance and total chl a concentration were lower in summer(0.78 × 108 ± SD 0.39 × 108 cell.m−2 and 59.0 ± SD 10.42 mgchla.m−2) than in winter (3.7 × 108 ± SD1.9 × 108 cell.m−2 and 106.64 ± SD 11.29 mgchla.m−2), coinciding with a high abundance of the gastropodPeringia ulvae in summer, which subsequently impacted 1st-cm chl a concentration by intense grazing. Boundand colloidal EPS carbohydrate temporal patterns were similar in winter (5.71 ± SD 3.95 and 4.67 ± SD3.45 μg.g−1, respectively) butwere different in summer (14.9±SD 4.05 and 5.60±SD 4.50 μg.g−1, respectively).Carbohydrate colloidal EPS appeared to be related to light and salinity, while 1st-mm chl a concentration wasnegatively affected by strong salinities and predation pressure by P. ulvae. The fluctuations of colloidal carbohydrateswere remarkably similar in the two seasons with peaks just after spring tideswhen the highest irradiancewas received by microphytobenthic cells. Apparently, colloidal EPS carbohydrates can protect cells against thehigh salinity values ranging from 32.3 to 50.4 PSU. The presence of bound EPS carbohydrates may be linked tosediment colonization and resistance of biofilm activity. Proteins in EPS were absent in winter and representeda small proportion in summer (10%), but they appeared to be a good indicator of potential synergistic effects betweenMPB and bacteria in summer. Conversely, bound EPS carbohydrates reached high levels in winter, whilethe number of bacteria decreased simultaneously, suggesting a negative effect on bacterial growth in the absenceof proteins in EPS. There was a lower proportion (31%) of low molecular weight EPS in summer than in winter(83%), possibly in relation to desiccation

Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France).

International audienceThe main goal of this work was to study the dynamics and biochemical composition of ExtraCellular Polysaccharides (ECPS), a fraction of the Extracellular Polymeric Substances (EPS) produced during the development of a microphytobenthic biofilm in a European intertidal mudflat (Marennes-Oléron Bay, France) during winter. Microphytobenthic biomass was surveyed during four consecutive emersion periods to confirm the biofilm growth. Bacteria abundance was also checked considering the importance of heterotrophic bacteria observed by various authors in the dynamics of EPS. Various colorimetric assays, coupled to biochemical chromatographic analysis were used to characterize the three main fractions of extracted EPS: colloidal, bound and residual. The monosaccharide distribution of colloidal ECPS highlighted their role of carbon source for bacteria (> 50 % of glucose) even if no increase of colloidal carbohydrate amounts was observed during the tidal exposure. Bound ECPS were composed of deoxy or specific sugars (30 % rhamnose) and uronic acids (18 % galacturonic acid). Their levels and dynamics could be correlated to the development of the microphytobenthic biofilm, enhancing the stabilization of the sediment or increasing binding forces accordingly. Residual fractions, containing refractory bound ECPS and other internal polymeric substances, were composed of various carbohydrates. The high ratio of glucose in these fractions (18 to 43 %) was interesting as it was once attributed to colloidal sugars due to poor extraction procedures. Finally, the presence of inositol (15 %) was significant since no author has highlighted it before, knowing that inositol is a major growth factor for heterotrophic bacteria