L’objectif de cette thèse est d’encapsuler dans une matrice polyosidique des probiotiques sous forme de biofilm en utilisant comme modèle Lactobacillus paracasei ATCC334. La matrice d’encapsulation (billes) est obtenue par gélification ionique d’une pectine amidée et faiblement méthylée en présence d’ions calcium. Nous avons remarqué que le réseau de pectine dans ces billes d’un diamètre d’environ 500 µm permet la formation de microcolonies sphériques (biofilm-like) avec une taille de 25 µm de diamètre. Une augmentation de la concentration bactérienne de L. paracasei ATCC334 de 3 Log d’UFC après 24 h de croissance des bactéries immobilisées est observée pour atteindre au sein de ces billes une concentration supérieure à 10 Log d’UFC/g humide. Nous avons également noté une distribution homogène des microcolonies dans les billes et une organisation structurée des bactéries au sein des microcolonies. En effet, une adhésion des bactéries à la matrice pectine est observée ainsi que la présence de substances polymériques permettant de lier les bactéries les unes aux autres. Les résultats suggèrent donc des phénomènes d’interaction entre le réseau formé par la pectine et les bactéries dans les billes. Nous avons montré que L. paracasei ATCC334 formulée en biofilm dans ces billes de pectine présente une résistance accrue à un stress gastrique (pH 2) et au séchage par lyophilisation. La capacité d’adhésion des bactéries formulées à des cellules épithéliales est conservée et la pectine semble stimuler cette adhésion aux cellules de l’hôte. Des résultats in-vivo utilisant un modèle murin d’inflammation intestinale montrent que les biofilms de L. paracasei ATCC334 sont libérés au niveau intestinal où ils s’implantent notamment dans le côlon. Des microcolonies de tailles approchant les 20 µm sont retrouvées au niveau du côlon suggérant que les billes de pectine ont libéré les bactéries sous forme de biofilm. Par ailleurs, l’administration aux souris de la formulation à base de pectine avec des biofilms de probiotiques a entrainé chez les souris ayant reçu un traitement DSS (une molécule capable de déclencher une inflammation intestinale) : une perte de poids moindre, un état de santé général amélioré, une muqueuse colique moins altérée ainsi qu’une diminution de la réponse inflammatoire. / The aim of this thesis is to encapsulate biofilm probiotics in a polysaccharide matrix using Lactobacillus paracasei ATCC334 as a model. The encapsulation matrix (beads) is obtained by the ionotropic gelation of amidated low-methoxylated pectin with calcium ions. We noticed that the pectin network in these beads with a diameter of approximately 500 microns, allow the formation of spherical microcolonies (biofilm-like) with a diameter of 25 microns. An increase of 3 Log CFU in the bacterial concentration of L. paracasei ATCC334 is observed after 24 hours of growth of the immobilized bacteria, while the observed concentration in these beads reaches more than 10 Log of CFU/g wet. We also noticed that the microcolonies within the beads are homogeneously distributed and the bacteria within the microcolonies are well structured. Moreover, a bacterial adhesion to the pectin matrix is observed as well as the presence of polymeric substances that bind the bacteria to each other. Our results suggest that interaction phenomenon may take place between pectin network and bacteria within beads. We also showed that biofilms of L. paracasei ATCC334 formulated in these pectin beads exhibit increased resistance to the gastric stress (pH 2) and to the freeze-drying process. In addition, the adhesion capacity of the formulated bacteria to epithelial cells is conserved and pectin seems to stimulate this adhesion to host cells. In-vivo results using a murine model presenting intestinal inflammation showed that L. paracasei ATCC334 biofilms are released in the intestinal level and are specifically implanted in the colon. Moreover, microcolonies of sizes approaching 20 μm are found in the colon suggesting that the bacteria are released in their biofilm form. In addition, the administration of pectinate beads containing probiotic biofilms to mice which have received a DSS treatment (inducing intestinal inflammation) resulted in: less weight loss of mice, improved their overall health status, less injured colonic mucosa and a decrease in the inflammatory response.

Ziel: In vorliegender In-situ-Studie sollte der Einfluss verschiedener fluorid- und zinnhaltiger Mundspüllösungen auf die Bildung des initialen bakteriellen Biofilms auf Schmelz-, Komposit- und Glasionomerzementproben untersucht werden. Die verwendeten Spüllösungen wurden einheitlich auf einen pH-Wert von 4,5 adjustiert. Untersucht wurden die initiale bakterielle Kolonisation und die Glucanbildung. Material und Methoden: Für die initiale Biofilmbildung wurden Probekörper aus bovinem Schmelz, Komposit und Glasionomerzement mittels individueller Schienen intraoral exponiert. Nach 1-minütiger oraler Applikation spülten die 6 Proband:innen für 1 min mit 10 ml der fluorid- und zinnionenhaltigen Mundspüllösungen. Die Spüllösungen enthielten Natriumfluorid, Natriummonofluorphosphat, Zinnfluorid und Aminfluorid mit einer Fluoridkonzentration von 500 ppm und Zinnchlorid mit einer Konzentration von 1563 ppm. Nach Applikation der Mundspüllösungen wurden die Schienen weitere 7 h 58 min getragen. Als Negativkontrolle wurden die Probekörper ohne Mundspülung für 8 h getragen. Zur Visualisierung und Quantifizierung der adhärenten Bakterien dienten die fluoreszenzmikroskopischen Verfahren DAPI, Concanavalin A und BacLightTM. Die statistische Auswertung erfolgte mittels Kruskal-Wallis- und Mann-Whitney-U-Test sowie der Bonferroni-Holm-Korrektur. Ergebnisse: Die initiale bakterielle Kolonisation konnte durch die Verwendung von zinnfluorid-, zinnchlorid- und aminfluoridhaltigen Mundspüllösungen auf den Schmelz-, Komposit- und Glasionomerzementproben signifikant reduziert werden. Die stärkste Reduktion der Gesamtzahl adhärenter Bakterien auf allen untersuchten Materialien war nach Spülung mit Zinnfluorid zu verzeichnen. Die Gesamtzahl adhärenter Mikroorganismen konnte im Vergleich zur Kontrolle (1,92*106 ± 7,64*105 Bakterien/cm²) durch die Anwendung von Zinnfluorid auf den Schmelzproben auf 3,54*105 ± 4,47*105 Bakterien/cm², auf Kompositproben auf 2,87*104 ± 3,25*104 Bakterien/cm² und auf Glasionomerzementproben auf 2,90*105 ± 1,62*105 Bakterien/cm² reduziert werden. Schlussfolgerungen: Zinnfluorid-, zinnchlorid- und aminfluoridhaltige Mundspüllösungen reduzieren die initiale bakterielle Biofilmbildung in situ auf Schmelz-, Komposit- und Glasionomerzement bei pH 4,5 am effektivsten. Zinnionenhaltige Mundspüllösungen inhibieren die bakterielle Kolonisation in situ auf Kompositprobekörpern. Zinnfluorid-, zinnchlorid- und aminfluoridhaltige Mundspüllösungen können zum oralen Biofilmmanagement empfohlen werden. Weitere In-situ- und In-vivo-Studien sind notwendig, um den Einfluss von Fluorid- und Zinnionen auf die Bildung der Pellikel und des initialen bakteriellen Biofilms auf dentalen Oberflächen zu untersuchen.

Diese Arbeit erlaubt einen erweiterten Einblick in die Regulation c-di-GMP-abhängiger Prozesse im Rahmen der Makrokolonieentwicklung von E. coli K12 AR3110, darunter die heterogene Expression des Masterregulators der Biofilmbildung CsgD und die räumlich differenzierte Matrixproduktion. Es konnte erstmals die Verteilung des Signalmoleküls c-di-GMP dargestellt werden und auch die damit einhergehe Dynamik der RdcA/B-DgcE-vermittelten Expression von csgD innerhalb einer Makrokolonie aufgeklärt werden. Zusätzlich konnten weitere Mechanismen in der Kontrolle der CsgD-Aktivität und Proteolyse identifiziert werden; darunter seine zelluläre Aggregation in spezifischen Zonen der Makrokolonie und sein proteolytischer Abbau (durch Lon-Proteasen). Diese Ergebnisse lassen eine präzise gesteuerte Koordination beschriebener Prozesse annehmen, die durch die Entstehung vertikaler Nähr- und Sauerstoffgradienten vermittelt wird. In ihrer Gesamtheit führen sie zu einer räumlich geordneten Produktion und Anordnung von Curlifasern und pEtN-Cellulose in der Makrokolonie, die zur Ausbildung der komplexen dreidimensionalen Matrixarchitektur führen. Jedes einzelne Matrixelement weist dabei eine charakteristische Zusammensetzung und Anordnung auf, die mit spezifischen Materialeigenschaften einhergehen. So bilden sie eine biofilminterne Gerüststruktur aus, die als mechanische Grundlage für die Auffaltung angenommen werden kann. Mikroskopische Analysen konnten matrixfreie, lokal proliferierende Zellen als treibende Kraft darstellen. Sie sind für den Aufbau und auch den Ausgleich eines Kompressionsdrucks verantwortlich, der die Auffaltung überhaupt verursacht. In ihrer Gesamtheit zeigt diese Arbeit, dass sowohl matrixfreie als auch matrixproduzierende Zellen eine horizontale Ausbreitung der Kolonie ermöglichen, ohne den Biofilm und darin befindliche Zellen zu beeinträchtigen. Auf diese Weise sind sie essenziell für die Aufrechterhaltung der physischen Integrität und Homöostase der Makrokolonie. / This work allows further insight into the regulation of c-di-GMP-dependent processes within the development of macrocolonies of E. coli K12 AR3110, including the heterogeneous expression of the master regulator CsgD and spatially differentiated matrix production. For the first time, the distribution of second messenger molecule c-di-GMP was visualized along the macrocolony axis. Furthermore, this study was able to elucidate the dynamics of RdcA/B-DgcE-controlled csgD expression and find additional mechanisms in the control of CsgD activity and proteolysis; including its cellular aggregation in specific zones and its proteolytic degradation by Lon-Preoteases. Overall, these results suggest a very precise coordination of cellular processes that depend on the presence of vertical oxygen and nutrient gradients which lead to a spatially defined production and arrangement of curlifibres as well as pEtN-Cellulose within the macrcocolony. This results in the formation of a complex three-dimensional matrix architecture with each individual matrix element exhibiting a characteristic composition and arrangement associated with specific material properties. That way they form a biofilm-internal scaffolding structure that represents the mechanical basis for macrocolony folding. Microscopic analyses were able to identify matrix-free and locally proliferating cells as driving force. They seem to be responsible for both creating as well as releasing pressure on matrix structures that causes buckling up in the first place. Taken together, this work shows that both matrix-free, proliferating cells and matrix-producing cells enable the horizontal spreading of the macrocolony without affecting the biofilm and cells located within. Thus, they are essential for maintaining physical integrity and homeostasis within the macrocolony.

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Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

Motivées par des normes de rejets en azote toujours plus sévères et par les besoins d'extension de certaines stations d'épuration, les agglomérations sont à la recherche de nouvelles technologies de traitement plus compactes et plus performantes. Dans ce sens, le procédé hybride, à lit fluidisé placé dans un réacteur de type boues activées (IFAS), est une nouvelle technologie de traitement du carbone et de l'azote très attractive. L'objectif de cette thèse est d'optimiser le dimensionnement du procédé IFAS en configuration trois bassins (anoxie/aérobie BA/aérobie IFAS) et d'apporter des recommandations sur la conduite du procédé (charge massique appliquée, température.). Pour cela, une double démarche expérimentale et numérique a été mise en place. Un pilote de 3 m3 alimenté en eau usée brute a été conçu, instrumenté et étudié pendant 2 ans au cours de 7 périodes stabilisées (entre 0,15 et 0,30 kgDBO5/kgMVSLM/j, température entre 10 et 22°C, et le séquençage de l'aération dans les bassins). La concentration en MES dans la liqueur mixte a été maintenue à 2,3 gMES/L et la concentration en oxygène entre 2 à 6 mgO2/L. Les capacités de nitrification du biofilm et de la liqueur mixte (NPRmax) ont été mesurées tous les 15 jours. Les performances d'élimination de l'azote (nitrification et dénitrification) et du carbone observées sont restées supérieur à 90% d'élimination pour une charge massique maximale de 0,30 kgDBO5/kgMVSLM/j entre 16 à 24°C. Le biofilm dispose d'une capacité de nitrification maximale de 0,90 gN/m2/j et tributaire des concentrations en oxygène dans la liqueur mixte (contraintes diffusionnelle). Le biofilm contribue en moyenne à hauteur de 60% du flux total nitrifié dans le réacteur IFAS pour des âges de boues < 5 jours à 16°C. La diminution du MLSRT en dessous de 4 jours a permis de limiter le développement des bactéries autotrophes dans la liqueur mixte (minimum 10% du flux total nitrifié par la liqueur mixte) mais pas de les supprimer totalement (apport de nitrifiante par détachement de biofilm) / Motivated by the increasingly demanding discharge consents and by the need to improve overall treatment capacity, water authorities are uninterruptedly examining better performing and more compact wastewater treatment technologies. Thanks to its compactness and to its capacity to treat both organic matter and nitrogen at an affordable cost, the IFAS process represents an attractive addition to improve retrofitting-activated sludge plants performance. The main objective of this thesis is to optimize IFAS process with regards to key operation parameters such as dimensioning, F/M ratio by combining experimental and mathematical modelling approaches. A 3 m3 pilot IFAS fed with raw wastewater was operated at the experimental hall of La Feyssine wastewater treatment plant, Villeurbanne, for a period of 2 years. The IFAS process was separated in 3 tanks to treat organic matter and total nitrogen separately (anoxic/aerobic, suspended/aerobic IFAS). The experimental study was divided in 7 periods with different steady state operation conditions each. The feasibility of nitrification at steady F/M ratios (between 0,1S to 0,30 kgBODS/kgMLVSS/d), at constant temperatures (between 10 - 22°C) and at different oxygen supply rates was investigated. TSS in mixed liquor were maintained at 2,3 gMLTSS/L and oxygen concentration between 2 to 6 mgO2/L. Biofilm mass and combined nitrification capacity of biofilm and mixed liquor (NPRmax) were measured on a weekly basis. The removal performance was up to 90% for nitrogen and carbon treatment with a maximal F/M ratio of 0,30 kgBODS/kgMLVSS/d between 16°C to 24 °C. The biofilm was able to nitrify 0,90 gN/m2/d (NPRmax) depending on the oxygen concentration in the mixed liquor (diffusional limitation). Under the operating conditions tested in this study, biofilm was responsible for 40 to 70% of NOx-N production in IFAS reactor during nitrification. Decreasing the MLSRT to less than 4 days limits the growth of autotrophic bacteria in the mixed liquor but does not halt it completely

This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.

Occurrence of organic micropollutants (OMPs) in aquatic environment is a worldwide concern. A long list of anthropogenic substances, including pharmaceuticals, hormones, etc., are frequently detected in natural water sources. Wastewater treatment plants are one main source of OMPs pollution, but also a key step to control OMPs dissemination into the environment. This dissertation focuses on the evaluation of Membrane Aerated Biofilm Reactor (MABR) as a sustainable process to treat wastewater polluted by OMPs. Furthermore, application of high intensity pulsed light is proposed as an innovative tertiary treatment to produce reclaimed water of high quality. In Chapter 1, a literature review was performed to investigate the occurrence and toxicity of 12 selected organic micropollutants (OMPs) in surface and ground water and the limitations of current available biological processes. Among these technologies, systems with enriched nitrifying activity were able to enhance the removal of specific OMPs through cometabolic activities. Thus, I proposed the use of a MABR with enriched nitrifying biomass to treat OMP polluted water. In Chapter 2, I studied the influence of biofilm thickness on the removal of 13 OMPs via MABR. Results demonstrated OMP removal was dependent on biofilm thickness and bacterial cell density. MABR demonstrated important efficiencies in the removal of ammonium, COD, acetaminophen and triclosan at early stages of biofilm thickness. However, the removal of nonpolar, hydrophobic 4 OMPs and anionic, acidic OMPs required thicker biofilms, achieving maximum removal at biofilm with 1.02 mm thickness and 2.2 × 106 cell mL-1. In Chapter 3, the contribution of sorption and biodegradation in the removal of OMPs via MABR was evaluated. At three stages of biofilm thickness studied, biodegradation dominated the removal for most OMPs. Heterotrophs played an important role in OMP biodegradation at all biofilm thickness, while autotrophic nitrifiers enhanced their contribution at thickness beyond 0.58 mm. Increased removal of pollutants like estrone and ethinyl estradiol were linked to the MABR enrichment with nitrifying bacteria. Sorption was essential for the removal of lipophilic and recalcitrant pollutants like triclosan. Finally, to provide high quality treated water for reuse, Chapter 4 explores the use of high-intensity pulsed light (HIPL) as post-treatment. The number of pulses and optical energy dose have a significant impact on the OMPs removal. HIPL demonstrated fast kinetics and efficient photodegradation – with significant OMPs removal within milliseconds. The findings from my Ph.D. dissertation indicate that MABR combined with high-intensity pulse light may be an effective treatment train for the efficient removal OMPs present in municipal wastewaters. This combined treatment process could potentially pave the way to produce high quality reclaimed water for various reuse purposes.