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Etude expérimentale de la formation des biofilms sous conditions hydrodynamiques contrôlées

Abstract : In the aquatic environment, 90% of microorganisms are present as a biofilm rather than free-swimming cells. Biofilms may develop on most of humid surfaces, in particular, in porous media for their high specific surface area. Biofilm formation in porous media is very interesting subject for many scientific researchers, because of its relevance to many industrial processes such as water treatment, soil bio- mediation, oil recovery and CO2 storage. However, the development of the biofilm is not just a passive aggregation of bacteria cells. It involves biological, physical and chemical interactions with the bacteria’s micro-environment. Several studies in macroscopic scale have shown that hydrodynamic conditions in porous media play an essential role on the dynamics of biofilm growth, which in turn affects hydrodynamic properties of porous media such as porosity, permeability and pressure drop. In this thesis we have developed an experimental device and an appropriate methodology for the characterization of biofilm’s structure. An experimental study on the influence of fluid flow on the formation and structure of biofilms was performed using a bacterial strain Pseudomonas putida. Biofilms were grown in micro Hele-Shaw flow cell (in PDMS or PMMA) under laminar flows (Re=0.04~2) and fed continuously with a nutrient medium. Characterization of initial colonization was also carried out in order to examine the statistical variability and reproducibility of experiments. Biofilm formation on a solid support under a sheared flow (Re=0.04 (0.0021 Pa) and Re = 2 (0.094 Pa)) was evaluated after 24, 48 and 72h of development. Observations were made under a confocal laser scanning microscopes using fluorescent tag. 2D images were taken at different positions in the flow cell and used to perform a 3D reconstruction of biofilm’s structure and an evaluation of its spatial distribution for an observation area of 12 *12mm². The results show that biofilms formation is not sensitive to initial colonization. A stratification of biofilm was also observed. The inner layer has a thin thickness (5~10 µm), but with a dense structure, while the outer layer show rather a filamentous structure. The ratio of volume fractions between these two layers varies from 3 to 12, depending on the formation time. This difference is more important in the case of low shear stress than that of high shear stress, which means that the upper part of the biofilm seems to be controlled by the hydrodynamic conditions. By analyzing the spatial distribution of the biomass, we found that after 48h, the biofilm present a significant heterogeneity and the volume fraction of biomass decreases after 72h for both two hydrodynamic conditions, which suggests probable detachments or erosions of biofilm. Concerning the growth kinetics, different apparent growth rates were observed for each observation time. These values are significantly below the growth rates observed in free culture medium. This result also indicates a possible effect of hydrodynamics on the growth of biofilm. This experimental study of biofilm formation in micro-scale allowed us to obtain the information on the biofilm structural and its apparent growth rate, as well as the hydrodynamic effect on its properties across several pores of the porous media. This scaling up makes it’s possible to develop eventually mathematical models to simulate the evolution biofilm’s morphology and its spatial distribution in the porous medium.
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Ana Cecilia de Andrade Pinho Medeiros. Etude expérimentale de la formation des biofilms sous conditions hydrodynamiques contrôlées. Mécanique des fluides [physics.class-ph]. Université Grenoble Alpes, 2016. Français. ⟨NNT : 2016GREAI011⟩. ⟨tel-01482919⟩

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