Protecting the quality and quantity of our water resources requires advanced treatment technologies capable of removing nutrients from wastewater. This research work investigated the capability of one such technology, a hollow-fiber membrane-aerated biofilm reactor (HFMBR), to achieve completely autotrophic nitrogen removal from a wastewater with high nitrogen content. Because the extent of oxygenation is a key parameter for controlling the metabolic processes that occur in a wastewater treatment system, the first part of the research investigated oxygen transfer characteristics of the HFMBR in clean water conditions and with actively growing biofilm. A mechanistic model for oxygen concentration and flux as a function of length along the non-porous membrane fibers that comprise the HFMBR was developed based on material properties and physical dimensions. This model reflects the diffusion mechanism of non-porous membranes; namely that oxygen follows a sorption-dissolution-diffusion mechanism. This is in contrast to microporous membranes in which oxygen is in the gas phase in the fiber pores up to the membrane surface, resulting in higher biofilm pore liquid dissolved oxygen concentrations. Compared to offgas oxygen analysis from the HFMBR while in operation with biofilm growing, the model overpredicted mass transfer by a factor of approximately 1.3. This was in contrast to empirical mass transfer coefficient-based methods, which were determined using either bulk aqueous phase dissolved oxygen (DO) concentration or the DO concentration at the membrane-liquid interface, measured with oxygen microsensors. The mass transfer coefficient determined with the DO measured at the interface was the best predictor of actual oxygen transfer under biofilm conditions, while the bulk liquid coefficient underpredicted by a factor of 3. The mechanistic model exhibited sensitivity to parameters such as the initial lumen oxygen concentration (at the entry to the fiber) and the diffusion coefficient and partitioning coefficients of oxygen in the silicone membrane material. The mechanistic model has several advantages over empirical-based methods. Namely, it does not require experimental determination of KL, it is relatively simple to solve without the use of advanced mathematical software, and it is based upon selection of the membrane-biofilm interfacial DO concentration. The last of these is of particular importance when designing and operating HFMBR systems with redox (aerobic/anoxic/anaerobic) stratification, because the DO concentration will determine the nature of the microenvironments, the microorganisms present, and the metabolisms that occur. During the second phase of the research, the coupling of two autotrophic metabolisms, partial nitrification to nitrite (nitritation) and anaerobic ammonium oxidation, was demonstrated in a single HFMBR. The system successfully treated a high-strength nitrogen wastewater intended to mimic a urine stream from such sources as extended space missions. For the last 250 days of operation, operating with an average oxygen to ammonia flux (J_O₂/J_NH₄⁺) of 3.0 resulted in an average nitrogen removal of 74%, with no external organic carbon added. Control of nitrite-oxidizing bacteria (NOB) presented a challenge that was addressed by maintaining the J_O₂/J_NH₄⁺ below the stoichiometric threshold for complete nitrification to nitrate (4.57 g O₂ / g NH₄⁺). The DO-limiting condition resulted in formation of harmful gaseous emissions of nitrogen oxides (NO, N2O), which could not be prevented by short-term control strategies. Controlling JO2/JNH4+ prevented NOB proliferation long enough to allow an anaerobic ammoniaoxidizing bacteria (AnaerAOB) population to develop and be retained for >250 days. Addition of a supplemental nutrient solution may have contributed to the growth of AnaerAOB by overcoming a possible micronutrient deficiency. Disappearance of the gaseous nitrogen oxide emissions coincided with the onset of anaerobic ammonium oxidation, demonstrating a benefit of coupling these two autotrophic metabolisms in one reactor. Obvious differences in biofilm density were evident across the biofilm depth, with a region of low density in the middle of the biofilm, suggesting that low cell density or exocellular polymeric substances were primarily present in this region, Microbial community analysis using fluorescence in situ hybridization (FISH) did not reveal consistent trends with respect to length along the fibers, but radial stratification of aerobic ammonia-oxidizing bacteria (AerAOB), NOB, and AnaerAOB were visible in biofilm section samples. AerAOB were largely found in the first 25% of the biofilm near the membrane, AnaerAOB were found in the outer 30%, and NOB were found most often in the mid-depth region of the biofilm. This community structure demonstrates the importance of oxygen availability as a determinant of how microbial groups spatially distribute within an HFMBR biofilm. The combination of these two aspects of the research, predictive oxygen transfer capability and the effect of oxygen control on performance and populations, provides a foundation for future application of HFMBR technology to a broad range of wastewaters and treatment scenarios. / Ph. D.

Most of microorganisms are ubiquitously able to live in both planktonic and biofilm states, which can be applied to dissolve the energy and environmental issues (e.g., producing biofuels and purifying waste water), but can also lead to serious public health problems. To better harness microorganisms, plenty of studies have been implemented to investigate the metabolism of planktonic and/or biofilm cells via multi-omics approaches (e.g., transcriptomics and proteomics analysis). However, these approaches are limited to provide the direct description of intracellular metabolism (e.g., metabolic fluxes) of microorganisms. Therefore, in this study, I have applied computational modeling approaches (i.e., 13C assisted pathway and flux analysis, flux balance analysis, and machine learning) to both planktonic and biofilm cells for better understanding intracellular metabolisms and providing valuable biological insights. First, I have summarized recent advances in synergizing 13C assisted pathway and flux analysis and metabolic engineering. Second, I have applied 13C assisted pathway and flux analysis to investigate the intracellular metabolisms of planktonic and biofilm cells. Various biological insights have been elucidated, including the metabolic responses under mixed stresses in the planktonic states, the metabolic rewiring in homogenous and heterologous chemical biosynthesis, key pathways of biofilm cells for electricity generation, and mechanisms behind the electricity generation. Third, I have developed a novel platform (i.e., omFBA) to integrate multi-omics data with flux balance analysis for accurate prediction of biological insights (e.g., key flux ratios) of both planktonic and biofilm cells. Fourth, I have designed a computational tool (i.e., CRISTINES) for the advanced genome editing tool (i.e., CRISPR-dCas9 system) to facilitate the sequence designs of guide RNA for programmable control of metabolic fluxes. Lastly, I have also accomplished several outreaches in metabolic engineering. In summary, during my Ph.D. training, I have systematically applied computational modeling approaches to investigate the microbial metabolisms in both planktonic and biofilm states. The biological findings and computational tools can be utilized to guide the scientists and engineers to derive more productive microorganisms via metabolic engineering and synthetic biology. In the future, I will apply 13C assisted pathway analysis to investigate the metabolism of pathogenic biofilm cells for reducing their antibiotic resistance. / Ph. D. / Most of microorganisms are ubiquitously able to live in both planktonic and biofilm states (i.e., floating in a flow and anchoring on a surface, respectively), which can be applied to dissolve the energy and environmental issues (e.g., producing biofuels and purifying waste water), but can also lead to serious public health problems (e.g., chronic infections). Therefore, deciphering the metabolism of both planktonic and biofilm cells are of great importance to better harness microorganism. Plenty of studies have been implemented to investigate the metabolism of planktonic and/or biofilm cells by measuring the abundances of single type of biological components (e.g., gene expression and proteins). However, these approaches are limited to provide the direct description of intracellular metabolism (e.g., enzyme activities) of microorganisms. Therefore, in this study, I have applied computational modeling approaches to both planktonic and biofilm cells for providing valuable biological insights (e.g., enzyme activities). The biological insights include 1) how planktonic cells response to mixed stresses (e.g., acids and organics) 2) how planktonic cells produce various chemicals, and 3) how biofilm cells generate electricity by rewiring the intracellular metabolic pathways. I also developed a novel platform to utilize multiple types of biological data for improving the prediction accuracy of biological insights of both planktonic and biofilm cells. In addition, I designed a computational tool to facilitate the sequence designs of an advanced genome editing tool for precisely controlling the corresponding enzyme activities. Lastly, I have also accomplished several outreaches in metabolic engineering. In summary, during my Ph.D. training, I have systematically applied computational modeling approaches to investigate the microbial metabolisms in both planktonic and biofilm states. The biological findings and computational tools can be utilized to guide the metabolic engineered to derive more productive microorganisms via metabolic engineering and synthetic biology. In the future, I plan to investigate how the pathogenic biofilm cells improve their antibiotic resistance and attempt to reduce such strong resistance.

[ES] Las biopelículas bacterianas y fúngicas contribuyen enormemente a la persistencia de muchas infecciones graves y potencialmente mortales, las cuales anualmente provocan millones de defunciones. Además, estas bacterias y hongos que crecen adheridas formando biopelículas son hasta 1.000 veces más resistentes a los tratamientos antimicrobianos convencionales, generando una carga económica significativa y dificultando su diagnóstico y tratamiento. Por tanto, es necesario buscar nuevas herramientas fiables para estudiar la dinámica de formación de biopelículas con el fin de mejorar las estrategias de tratamiento.El objetivo general de la tesis doctoral es la puesta a punto de un sistema basado en medidas de impedancia eléctrica para el estudio de la formación y dinámica de crecimiento de las biopelículas bacterianas (gram-positivas y gram-negativas) y fúngicas, así como de biopelículas complejas multi-especie como las de la placa dental subgingival de muestras periodontales humanas. Tras la puesta a punto del sistema, los objetivos específicos de la tesis doctoral son su aplicación como herramienta en la identificación de tratamientos efectivos contra biopelículas persistentes, la búsqueda de nuevos compuestos antimicrobianos con actividad anti-biofilm, así como la evaluación de novedosas nanopartículas autopropulsadas para la erradicación de biofilms multirresistentes. Finalmente, se ha evaluado su aplicación clínica directa en la selección de la terapia antibiótica para el tratamiento personalizado de pacientes con enfermedad periodontal. / [CA] Les biopelícules bacterianes i fúngiques contribueixen en gran manera a la persistència de moltes infecciones greus i potencialment mortals les quals provoquen anualment milions de morts. A més, estes bactèries i fongs que creixen adherides en forma de biopelícules son fins a 1000 vegades més resistents als tractaments antimicrobians convencionals, generant una càrrega econòmica significativa i dificultant el diagnòstic i tractament. Per això, es necessari trobar noves eines fiables per a estudiar la dinàmica de formació de biopelícules amb l'objectiu de millorar les estratègies de tractament. El objectiu general de la tesis doctoral es la posta a punt de un sistema basat en mesures d'impedància elèctrica per al estudi de la formació i dinàmica de creixement de les biopelícules bacterianes (gram-positives i gram-negatives) i fúngiques, així com de biopelícules complexes mutiespècie com les de la placa dental subgingival de mostres periodontals humanes. Una vegada posat a punt el sistema, els objectius específics de la tesis doctoral son la aplicació com a eina de la identificació de tractaments efectius contra biopelícules persistents, la recerca de nous compostos antimicrobians amb activitat antibiopelícula, així com la avaluació de noves nanopartícules autopropulsades per a l'eliminació de biofilms multiresistents. Finalment, s'ha avaluat l'aplicació clínica directa en la selecció de la teràpia antibiòtica per al tractament personalitzat de pacients amb periodontitis. / [EN] Bacterial and fungal biofilms contribute enormously to the persistence of many life-threatening infections, causing millions of deaths annually. In addition, bacteria and fungi growing as biofilms are up to 1.000 times more resistant to conventional antimicrobial treatments, resulting in a significant economic burden and challenging diagnosis and treatment. Therefore, there is a need to search for new reliable tools to study biofilm formation dynamics to improve treatment strategies. This doctoral thesis aims to set up an impedance-based system to study biofilm formation and dynamics of bacterial (gram-positive and gram-negative) and fungal species, as well as complex multi-species biofilms such as subgingival plaque collected from patients with chronic periodontitis. After the impedance system is set up, the specific objectives of the doctoral thesis are its application as a tool in the identification of effective treatment against persistent biofilms, testing new antimicrobial and anti-biofilm compounds, and the evaluation of novel self-propelled nanoparticles on the eradication of multi-resistant S. aureus biofilms. Finally, a clinical application of the impedance system is proposed, aiming at determining the best individual antibiotic therapy in dental clinics (personalized use of antibiotics). / Work performed at Genomics & Health Department at FISABIO Foundation and described in this doctoral thesis was supported by the Spanish Ministry of Science, Innovation and Universities scholarship FPU17/01302 to Miglė Žiemytė and a grant RTI2018-102032-B-I00 to Alex Mira Obrador. / Ziemyte, M. (2023). Towards Personalized Medicine in Antibiotic Treatment: Development of a Real-Time Cell Analysis System for Biofilm Studies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195434

Mathematical models of a nitritation: anaerobic ammonia oxidation (anammox)-coupled biofilm in a counter-diffusion hollow fiber membrane bioreactor (HFMBR) and a nitritation: anammox-coupled biofilm in a co-diffusion rotating biological contactor (RBC) were developed and implemented using AQUASIM. Four different start-up scenarios on the nitritation: anammox-coupled biofilm in an HFMBR were investigated. The supply of oxygen was simulated with the flow through the lumen of the hollow fiber membrane. For the four scenarios, two scenarios investigated the start-up when nitrite was supplied in the feed while the other two scenarios investigated when the source of nitrite was through nitritation only. The results showed that the presence of nitrite in the feed facilitated the start-up of the reactor. In addition, the results also showed that increasing oxygen flux through the membrane up to a certain ratio of ammonia flux with oxygen flux affected reactor performance by improving nitrogen removal and reducing start up time. For the nitritation: anammox-coupled biofilm in an RBC, four different process options were investigated: the number of reactors, the initial anammox (AnAOB) biomass fraction, the bulk oxygen concentration and the maximum biofilm thickness. Modeling results revealed that the steady state total nitrogen removal in RBC reactors in series occurred primarily in the first and second reactors. It is concluded that the number of reactors in series dictates the effluent performance and, therefore, this number can be selected depending upon the desired total nitrogen removal. Simulation results also revealed that increasing the initial AnAOB biomass fraction from 0.01% to 1.0% had no effect in the steady state nitrogen removal but had an effect in the required time to reach the steady state total nitrogen removal and the maximum biofilm thickness. Modeling results of the third process option showed that increasing the bulk oxygen concentration in the reactor from 0.2 g/m3 to 5 g/m3 linearly increased the steady state total nitrogen removal and reduced the time to reach the maximum biofilm thickness. Beyond 5 g/m3, steady state total nitrogen removal decreased. In addition, simulation results revealed that the thicker biofilm clearly showed a more linear correlation between the increase in bulk oxygen concentration and the increase in the steady state total nitrogen removal within a range of bulk oxygen concentrations. The results showed that RBC performance could be controlled by several process options: the number of reactors in series, initial biomass fraction, the bulk oxygen concentration and the maximum biofilm thickness. The mathematical modeling results for the HFMBR and RBC have shown that both have potential as carriers for nitritation: anammox-coupled biofilms targeted at the removal of nitrogen in the wastewater. / Master of Science

<p dir="ltr">This thesis explores the complex relationship between <i>Listeria</i> <i>monocytogenes</i> and food processing environments (FPEs), focusing on the persistence mechanisms of this pathogen despite rigorous sanitation efforts. <i>L</i>. <i>monocytogenes</i> is a significant public health concern due to its association with high mortality rates in vulnerable populations. Recent studies, including the first chapter of this thesis, have highlighted the challenges in eradicating this pathogen from FPEs, suggesting that biofilms play a crucial role in its persistence. Despite various strategies and enhanced sanitation protocols, the eradication of <i>L</i>. <i>monocytogenes</i> remains elusive, underlining the need for a deeper understanding of its biofilm-associated resilience. Chapter two synthesizes findings from a systematic review and meta-analysis of studies examining the microbial communities on FPE surfaces through metagenomics, aiming to uncover patterns that might explain <i>Listeria</i>'s long-term survival. In chapter three, the thesis delves into the nuanced role of biofilm composition and microbial diversity as factors enabling the persistence of <i>Listeria monocytogenes</i> in food processing environments (FPEs), despite comprehensive sanitation efforts. This section builds on the understanding that <i>L. monocytogenes</i> does not inherently form robust biofilms but thrives within complex microbial communities present on FPE surfaces. The findings presented in this thesis contribute to a more profound comprehension of <i>L. monocytogenes</i>' survival strategies, proposing a shift in focus towards the microbial ecosystem's metabolic interplays for the development of targeted control measures. This approach not only opens new avenues for research but also suggests practical implications for enhancing food safety protocols by considering the broader microbial dynamics at play within biofilms on FPE surfaces.</p>

Les doublets géothermiques sur aquifères superficiels jouent un rôle important en France pour le chauffage, le rafraichissement et la production d’eau chaude sanitaire des bâtiments. La pérennité de ces installations est cependant conditionnée par la possibilité de pouvoir garantir dans le temps la production puis la réinjection de l’eau souterraine. Le colmatage de la boucle géothermale est un problème majeur qui affecte de nombreuses installations sur l’ensemble du territoire français et remet en cause leur viabilité technico-économique. La compréhension et la prédiction de ce phénomène nécessite de nouveaux moyens de caractérisation et de modélisation des processus biogéochimiques couplés au fonctionnement des boucles géothermales dans des environnements de subsurface hétérogènes. Cette thèse a ainsi fait l’objet d’une nouvelle collaboration entre le laboratoire Géosciences Rennes et l’entreprise Antea group pour identifier les mécanismes à l’origine de ces phénomènes de colmatage et d’en caractériser les paramètres influents dans le but de construire un outil d’analyse des risques permettant l’anticipation de ces processus de colmatage. Le premier volet de la thèse expose la synthèse des typologies de colmatage (biogéochimique, chimique, biologique et physique). Cette synthèse est issue de l’étude de la littérature sur la problématique de colmatage des forages d’eau et des retours d’expérience concernant les doublets géothermiques recensés et étudiés durant cette thèse. Le second volet présente les inventaires régionaux des problématiques de colmatage recensées en France dans différents contextes hydrogéologiques. Ces inventaires ont permis de délivrer un état des lieux à grande échelle de la problématique de colmatage et d’étudier les contextes associés à chaque type de colmatage. Dans le troisième volet, de nouvelles méthodologies de caractérisation in-situ des phénomènes de colmatage biogéochimique liés à l’oxydation du manganèse et du fer sont présentées. Elles ont été développées dans le cadre de ces travaux de thèse à partir des investigations menées sur différents sites confrontés à des problèmes d’exploitation. Ces méthodologies d’investigations pluridisciplinaires couplent la mesure des propriétés hydrauliques, des concentrations en éléments chimiques et de la diversité microbienne afin d’identifier la problématique impactant le fonctionnement de l’installation et d’en définir in fine les causes. Le quatrième volet expose les résultats d’une campagne de terrain effectuée sur un doublet géothermique impacté par un processus de colmatage biogéochimique. Cette campagne a permis d’identifier les composantes clés de la réactivité biochimique impliquée dans le colmatage : distribution du flux, hétérogénéité chimique et diversité microbiologique. A l’aide du code de calcul PHREEQC, un modèle géochimique simulant les cinétiques de précipitation observées a été développé. Il permet l’étude quantitative des mécanismes biogéochimiques favorisant l’apparition rapide du colmatage. Enfin, les retours d’expériences de l’exploitation des doublets sur nappes superficielles ont démontré le besoin d’une méthodologie fiable d’analyse des risques, permettant d’anticiper l’apparition des processus de colmatage à chaque étape de la vie du projet. Ainsi, à partir de la synthèse de la littérature scientifique et technique et des conclusions des études menées durant cette thèse, les facteurs de risques d’apparition des phénomènes de colmatage ont été déterminés. L’analyse des risques de colmatage intégrant ces différents facteurs a été implémentée à travers l’élaboration de méthodes développées sous Python 3. L’outil ARCADE (Analyse des Risques de Colmatage et Aide à la Décision) a été conçu d’une part pour évaluer le risque et d’autre part pour informer l’utilisateur averti des bonnes pratiques et moyens préventifs. Ces bonnes pratiques d’analyse et de gestion préventive sont présentées dans le dernier volet de cette thèse. / The sustainability of geothermal systems using shallow aquifers for heating, cooling and hot water production depends on the possibility to ensure, over long time-scales, the production and the reinjection of groundwater in the aquifer. Clogging of the geothermal loop is a major issue affecting the technical and economic viabilities of numerous operations in France. The understanding and prediction of this phenomenon requires new methods of characterization and modelling of biogeochemical processes coupled to the operation of geothermal loops in heterogeneous subsurface environments. This thesis is thus the result of a new collaboration between the Géosciences Rennes lab and Antea group to identify the mechanisms at the origin of clogging phenomena and characterize their controlling parameters, in order to establish a risk assessment tool allowing the anticipation of clogging processes. The first part of the thesis describes the main clogging processes (biogeochemical, chemical, biological and physical). This synthesis is the result of (1) the study of the literature dealing with the clogging of water wells and (2) our feedbacks on the geothermal doublets identified and studied during this thesis. The second part presents the regional inventories of shallow geothermal systems impacted by clogging problems identified in different hydrogeological contexts in France. These inventories provide a large-scale perspective of clogging phenomena and allow to study the contexts associated with each type of clogging processes. In the third part, new methodologies for the in-situ characterization of biogeochemical clogging phenomena linked to manganese and iron oxidation are presented through case studies of sites affected by clogging issues. These interdisciplinary studies couple the measurement of hydraulic properties, chemical element concentrations and bacterial diversity, to identify the specific issue impacting the operation and to define its causes. The fourth part presents the results of an interdisciplinary field campaign carried out on a geothermal doublet impacted by a biogeochemical clogging process. This campaign documented the key components involved in mixing induced biogeochemical reactivity: flow distribution, chemical heterogeneity and microbiological diversity. Using PHREEQC, a geochemical model simulating observed kinetics of precipitation was developed in order to quantitatively explore the biogeochemical mechanisms favoring rapid clogging. Feedback from shallow geothermal systems operation has demonstrated the need for a reliable risk analysis methodology that allowed to anticipate the apparition of clogging processes at each stage of the project life (part 5). From the synthesis of the scientific & technical literature and the conclusions of the studies carried out during this thesis, the risk factors for the appearance of clogging phenomena were determined. A clogging risk analysis integrating these factors was implemented through the development of methods developed under Python 3. The methodology of the ARCADE tool (Analyse des Risques de Colmatage et Aide à la Décision) is designed to assess the risk and to inform users of good practices and preventive methods. These good practices for analysis and preventive methods are presented in the last part of this thesis.

Les composés organiques hydrophobes (HOC), une grande famille de molécules naturelles ou d’origine anthropique incluant les lipides et les hydrocarbures, constituent une part significative de la matière organique dans les écosystèmes marins. Du fait de leur faible solubilité dans l’eau, les bactéries qui les dégradent requièrent la mise en place de fonctions cellulaires spécifiques permettant d’augmenter la fraction assimilable de ces HOC. La formation de biofilms à l’interface eau-HOC est une de ces stratégies adaptatives. C’est le cas pour Marinobacter hydrocarbonoclasticus SP17, modèle d’étude utilisé au laboratoire, qui est capable de former des biofilms sur un large spectre de HOC métabolisables tels que les alcanes, les triglycérides et les alcools gras. Le but de mes recherches consistait à améliorer la compréhension du processus d’adhésion et de développement des biofilms sur les HOC, à travers la caractérisation fonctionnelle de 10 gènes candidats mis en évidence lors d’analyses d’expression en protéomique et en transcriptomique. Pour mener à bien ce projet, des outils génétiques et une caractérisation fonctionnelle propre à chaque gène ont dû être développés. L’étude fonctionnelle du gène MARHY2686 a relevé son implication dans la formation de biofilm sur les alcanes. La co-expression de MARHY2686 et des gènes adjacents MARHY2687 et MARHY2685 en transcriptomique, leur distribution phylogénétique et leur conservation de la synthénie suggèreraient que ces trois gènes soient impliqués dans le même processus biologique. D’après l’identité forte de 36 % qui existe entre la protéine MARHY2686 et une protéine périplasmique AdeT d’un système de pompe d’efflux tripartite d’Acinetobacter baumanii, cette protéine, en association avec MARHY2687 et MARHY2685, pourrait faire partie d’un système de ce type. Par ailleurs, des observations ont permis d’envisager une implication potentielle de ce gène dans l’assimilation des HOC ou dans l’accumulation des réserves lipidiques intracellulaires. M. hydrocarbonoclasticus SP17 utilise les pili de type IV lors de la formation de biofilm sur les HOC. Ces appendices interviennent lors de l’adhésion de cette souche à des HOC ainsi que dans un processus de détachement d’un support hydrophobe. Les pili pourraient soit intervenir directement pour permettre à la bactérie de se détacher de la surface à laquelle elle s’est adhérée, soit indirectement par l’action de bactériophages. La présence d’une mobilité de type twitching sur les HOC a pu être également envisagée. Enfin, le rôle du système de sécrétion de type VI (T6SS), connu pour permettre à la bactérie d’interagir avec une cellule hôte, lors de la formation de biofilm mono-spécifique sur HOC, où aucun autre microorganisme que M. hydrocarbonoclasticus SP17 n’est présent, a été étudié. / Hydrophobic organic compounds (HOC), a large family of naturally-produced or anthropogenic molecules including lipids and hydrocarbons, represent a significant part of organic matter in marine ecosystems. Because of their low solubility in water, bacteria that degrade those compounds require the establishment of specific cell functions to increase their biodisponibility. Biofilm formation in water-HOC interface is one of these adaptations. The model of bacteria used in our laboratory, Marinobacter hydrocarbonoclasticus SP17, is able to form a biofilm on a wide range of HOC, such as alkanes, fatty alcohols and triglycerides, in order to use them as a carbon and energy source. The main purpose of my work was to broaden the knowledge of how bacteria adhere to and from biofilms on HOC, through the functional characterization of 10 candidate genes highlighted during proteomic and transcriptomic studies. Genetic tools and a gene-specific functional characterization have been developed in order to carry out this project. Functional study conducted on MARHY2686 revealed its involvement in the formation of biofilm on alkanes. Co-expression of MARHY2686 and the adjacent genes MARHY2687 and MARHY2685 durnig transcriptomic analysis together with their phylogenetic distribution and synteny conservation suggest that these three genes are involved in the same biological process. According to the high peptide sequence identity between MARHY2686 and AdeT, a periplasmic protein of a tripartite efflux pump system of Acinetobacter baumanii, MARHY2686 in combination with MARHY2687 and MARHY2685 could be the components of such a system. Other phenotypic observations would consider the involvement of MARHY2686 either in the assimilation of HOC or in the accumulation of intracellular lipid reserves. M. hydrocarbonoclasticus SP17 uses type IV pili during biofilm formation on HOC. These appendages are involved in the adhesion of this strain to and in a detachment process from HOC. Type IV pili could either act directly to allow bacteria to detach from the surface to which it is adhered, or indirectly through the action of bacteriophages. The presence of twitching motility on HOC has also been suggested. Finally, the role of the type VI secretion system (T6SS), a well-known protein system which allows interactions between bacteria and host cells, during the formation of a mono-species biofilm on HOC where no other microorganism than M. hydrocarbonoclasticus SP17 is present, has been studied.

Chapter–I Introduction Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are essentially non-linear waves that propagate at supersonic speeds. Such disturbances occur in steady transonic or supersonic flows, during explosions, earthquakes, tsunamis, lightening strokes and contact surfaces in laboratory devices. Any sudden release of energy (within few μs) will invariably result in the formation of shock wave since it is one of the efficient mechanisms of energy dissipation observed in nature. The dissipation of mechanical, nuclear, chemical, and electrical energy in a limited space will result in the formation of a shock wave. However, it is possible to generate micro-shock waves in laboratory using different methods including controlled explosions. One of the unique features of shock wave propagation in any medium (solid, liquid or gases) is their ability to instantaneously enhance pressure and temperature of the medium. Shock waves have been successfully used for disintegrating kidney stones, non-invasive angiogenic therapy and osteoporosis treatment. In this study, we have generated a novel method to produce micro-shock waves using micro-explosions. Different biological applications were developed by further exploring the physical properties of shock waves. Chapter – II Bacterial transformation using micro-shock waves In bacteria, uptake of DNA occurs naturally by transformation, transduction and conjugation. The most widely used methods for artificial bacterial transformation are procedures based on CaCl2 treatment and electroporation. In this chapter, controlled micro-shock waves were harnessed to develop a unique bacterial transformation method. The conditions have been optimized for the maximum transformation efficiency in E. coli. The highest transformation efficiency achieved (1 × 10-5 transformants per cell) was at least 10 times greater than the previously reported ultrasound mediated transformation (1 × 10-6 transformants per cell). This method has also been successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella Typhimurium. This novel method of transformation has been shown to be as efficient as electroporation with the added advantage of better recovery of cells, economical (40 times cheaper than commercial electroporator) and growth-phase independent transformation. Chapter – III Needle-less vaccine delivery using micro-shock waves Utilizing the instantaneous mechanical impulse generated behind the micro-shock wave during controlled explosion, a novel non-intrusive needleless vaccine delivery system has been developed. It is well established, that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle free devices for epidermal delivery have inherent problems from the perspective of patient safety and comfort. The penetration depth of less than 100 µm in the skin can elicit higher immune response without any pain. Here the efficient utilization of the device for micro-shock wave mediated vaccination was demonstrated. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. The device mediated vaccination elicits better protection as well as IgG response even in lower vaccine dose (ten-fold lesser), compare to other routes of vaccination. Chapter – IV In vitro and in vivo biofilm disruption using shock waves Many of the bacteria secrete highly hydrated framework of extracellular polymer matrix on encountering suitable substrates and get embedded within the matrix to form biofilm. Bacterial colonization in biofilm form is observed in most of the medical devices as well as during infections. Since these bacteria are protected by the polymeric matrix, antibiotic concentration of more than 1000 times of the MIC is required to treat these infections. Active research is being undertaken to develop antibacterial coated medical implants to prevent the formation of biofilm. Here, a novel strategy to treat biofilm colonization in medical devices and infectious conditions by employing shock waves was developed. Micro-shock waves assisted disintegration of Salmonella, Pseudomonas and Staphylococcus biofilm in urinary catheters was demonstrated. The biofilm treated with micro-shock waves became susceptible to antibiotics, whereas the untreated was resistant. Apart from medical devices, the study was extended to Pseudomonas lung infection model in mice. Mice exposed to shock waves responded well to ciprofloxacin while ciprofloxacin alone could not rescue the mice from infection. All the mice survived when antibiotic treatment was provided along with shock wave exposure. These results clearly demonstrate that shock waves can be used along with antibiotic treatment to tackle chronic conditions resulting from biofilm formation in medical devices as well as biological infections. Chapter – V Shock wave responsive drug delivery system for therapeutic application Different systems have been used for more efficient drug delivery as well as targeted delivery. Responsive drug delivery systems have also been developed where different stimuli (pH, temperature, ultrasound etc.) are used to trigger the drug release. In this study, a novel drug delivery system which responds to shock waves was developed. Spermidine and dextran sulfate was used to develop the microcapsules using layer by layer method. Ciprofloxacin was loaded in the capsules and we have used shock waves to release the drug. Only 10% of the drug was released in 24 h at pH 7.4, whereas 20% of the drug was released immediately after the particles were exposed to shock waves. Almost 90% of the drug release was observed when the particles were exposed to shock waves 5 times. Since shock waves can be used to induce angiogenesis and wound healing, Staphylococcus aureus skin infection model was used to show the effectiveness of the delivery system. The results show that shock wave can be used to trigger the drug release and can be used to treat the wound effectively. A brief summary of the studies that does not directly deal with the biological applications of shock waves are included in the Appendix. Different drug delivery systems were developed to check their effect in Salmonella infection as well as cancer. It was shown for the first time that silver nanoparticles interact with serum proteins and hence the antimicrobial properties are affected. In a nutshell, the potential of shock waves was harnessed to develop novel experimental tools/technologies that transcend the traditional boundaries of basic science and engineering.

L'assainissement non collectif concerne 12 à 15 millions de personnes en France. La filière classique de ce mode d’assainissement se compose généralement d'un prétraitement anaérobie par une fosse septique recevant l’ensemble des eaux usées domestiques suivi d’un système d’infiltration dans le sol ou d’un filtre à sable. Le filtre à sable vertical drainé met à profit le pourvoir épuratoire qui est principalement lié à la présence d’une biomasse sous forme d’un biofilm. Cette dynamique de la croissance de la biomasse ou du biofilm est soumise à l’impact de la nature de matériaux filtrants. L’écoulement insaturé dans ces systèmes conditionne également cette croissance du biofilm. Dans ce contexte, l'objectif du travail de la thèse est d’appréhender les mécanismes mis en jeu et particulièrement l’impact des matériaux dans le fonctionnement des filtres en comparant notamment deux types de matériaux: les sables de rivière et les agrégats concassés. Pour cela, une étude expérimental sur une unité pilote composé des réacteurs de filtration du diamètre de 30cm et différents épaisseurs de garnissage (15, 30 et 70cm) a été construite. Les réacteurs garnis de deux sables roulés et deux agrégats concassés, sont alimentés en effluent septique avec une charge volumique 12cm/jour par 10 bâchés par jour. Suite des matériaux, une étude de la performance épuratoire avec le suivi des composants biochimiques de la biomasse totale et de la matrice extracellulaire du biofilm est réalisée en comparant notamment les deux types de matériaux filtrants. / The onsite wastewater treatment systems concern 12 to 15 million of people in France. The treatment plant is generally composed by a septic tank as pretreatment, followed by soil infiltration field or sand filtration bed. The vertical drained sand filter provides the purification capacity thanks to the presence of a biomass in form of the biofilm. The dynamic of the biomass growth or the biofilm development is under the impact of filter materials’ nature. In this context, the objective of this work is to understand the mechanisms involved and especially the impact of medium in the functioning of the filtration reactor by comparing two types of packing materials: river sands and crushed aggregates. For this purpose, an experimental study is conducted with pilot unity composed by filtration reactors of 30cm of diameter and different packing thicknesses (15, 30 and 70cm). The reactors packed with two river sands and two crushed aggregates are fed with septic effluent with a volumic hydraulic charge of 12cm/day by 10 batches per day. Based on a characterization of materials, a study of purification performance with biochemical components monitoring of the total biomass and the extracellular matrix of the biofilm is realized by comparing two types of filter materials. The purification performance has presented similar efficiencies of particulates and organic matters removals by fine river sand and fine crushed aggregate. The nitrogen pollutants removals are more effective in the fine river sand which presents the finest granulometry with an alternative of aerobic and anoxic phases along the reactor depth and with a biomass more abundant. The distribution and the quality of the total biomass and the extracellular matrix differentiate between the river sand and the crushed aggregates. As the reference material, the fine river sand presented an earlier stabilization of total biomass growth with a less important production of extracellular exudates compared to the crushed aggregates. The origin of impacts brought by the crushed aggregates might be due to the higher fine particles content which created microenvironments poor in substrates or in oxygen and also due to a more heterogeneous mineralogy. The extracellular components of highest percentage in the biofilm of crushed aggregate are polysaccharides type substances.

Le principal objectif de cette thèse était de mieux comprendre l’importance des Substances Polymériques Extracellulaires (SPE) dans la structuration et la formation des biofilms benthiques ; tout en s’inscrivant dans une étude plus globale des mécanismes écologiques impliqués dans le fonctionnement des vasières intertidales. La mise au point des dosages biochimiques a été effectuée sur le mucilage de l’algue Chaetomorpha aerea et a permis en parallèle de purifier un polysaccharide sulfaté riche en galactose, présentant une activité bactéricide sélective contre la souche Staphylococcus aureus (ATCC 25923). Les études biochimiques et écologiques menées sur les SPE extraits de la vasière charentaise ont ensuite permis de quantifier leur dynamique de production et leur composition, en fonction des conditions environnementales. La présence de désoxy-sucres et d’acides uroniques au sein des SPE capsulaires a laissé supposer que ces fractions jouaient un rôle important dans la formation et le devenir du biofilm microphytobenthique. La dernière partie des travaux a permis de caractériser les propriétés acide/base de Lewis et hydrophile/hydrophobe de la surface de la micro-algue Navicula jeffreyi, impliquée dans la formation de biofilms benthiques, par des méthodes classiques d’analyse. L’utilisation d’une nouvelle méthode, la Chromatographie Gazeuse Inverse (CGI), a permis d’obtenir des résultats intéressants et relativement similaires, confirmant le caractère prometteur de la CGI pour l’étude des propriétés de surface des micro-organismes. / The main goal of this thesis was to better understand the importance of Extracellular Polymeric Substances (EPS) in the structuring and formation of benthic biofilms; while considering a global conception of the ecological mechanisms involved in the functioning of intertidal mudflats. The development of the biochemical assays was done on the mucilage of the macroalgae Chaetomorpha aerea and allowed purifying a polysaccharide rich in galactose, showing a selective bactericidal activity against Staphylococcus aureus (ATCC 25923). Then, the biochemical and ecological studies concerning the EPS extracted from the local mudflat allowed studying their dynamic of production and composition in relation to environmental conditions. The presence of deoxy sugars and uronic acids in the bound EPS highlighted their important roles during the formation and the life of microphytobenthic biofilms. The last part of the work was used to characterize the acid/base of Lewis and hydrophilic/hydrophobic surface properties of the microalgae Navicula jeffreyi, involved in the formation of benthic biofilms, by using classical analysis methods. The use of a new method, named Inverse Gas Chromatography (IGC), allowed getting interesting and relatively similar results, confirming the potential of the method to study the surface properties of microorganisms.