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Thèse A. Larue

6 avril 2018

Experimental Methodologies to Explore 3D Development of Biofilms in Porous Media

Soutenance de thèse Anne Larue

Mardi 27 mars à 9 h 30 - Centre d’Enseignement et de Congrès Hopital Purpan, salle 14 (330 Avenue de Grande Bretagne, 31300 Toulouse.)

Abstract :

Biofilms are microbial communities developing at the interface between two phases, usually solid- liquid, where the micro-organisms are nested in a self-secreted polymer matrix. The biofilm mode of growth is predominant in nature (for e.g. the slimy matter forming on rocks at river bottoms, the viscous deposit in water pipes or even dental plaque) and confers a suitable environment for the development of the micro-organisms. This is particularly the case for porous media which provide favourable substrates given their significant surface to volume ratio. The multi-physical framework of biofilms in porous media is highly complex where the mechanical, chemical and biological aspects interacting at different scales are poorly understood and very partially controlled. An example is the feedback mechanism between flow, spatial distribution of the micro-organisms and the transport of nutrient (by diffusion and advection). Biofilms developing in porous media are a key process of many engineering applications, for example biofilters, soil bio-remediation, CO2 storage and medical issues like orthopaedic infections.
Progress in this domain is substantially hindered by the limitations of experimental techniques in metrology and imaging in opaques structures. The main objective of this thesis is to propose robust and reproducible experimental methodologies for the investigation of biofilms in porous media. An experimental workbench under controlled physical and biological conditions is proposed along with a validated 3D imaging protocol based on X-ray micro-tomography (XR MT) using a novel contrast agent (barium sulfate and agarose gel) to quantify the spatial distribution of the biofilm.

At first, the XR MT-based methodology is compared to a commonly used techniques for biofilm observation : one or multiple photon excitation fluorescence microscopy, here two-photon laser scanning microscopy (TPLSM). This comparison is performed on Pseudomonas Aeruginosa biofilms grown in transparent glass capillaries which allows for the use of both imaging modalities. Then, the study of uncertainty associated to different metrics namely volume, 3D surface area and thickness, is achieved via an imaging phantom and three different segmentation algorithms. The quantitative analysis show that the protocol enables a visualisation of the biofilm with an uncertainty of approximately 17% which is comparable to TPLSM (14%). The reproducibility and robustness of the XR MT-based methodology is demonstrated.
The last step of this work is the achievement of a novel bioreactor elaborated by additive manufacturing and controlled by a high-performance micro-fluidic system. The experimental workbench that we have designed enables to monitor in real-time the evolution of transport properties (effective permeability), O2 concentrations and biofilm detachment by spectrophotometry, all under controlled hydrodynamical conditions. Our methodology allows to investigate the influence of biophysical parameters on the colonisation of the porous medium, for example, the influence of flow rate or nutrient concentration on the temporal development of the biofilm.

In conclusion, the thesis work proposes a robust and reproductible experimental methodology for the controlled growth and 3D imaging of biofilms in porous media ; while providing versatility in the control of the substrate’s micro-architecture as well as on the flow and biochemical culture conditions. To our knowledge, the scientific approach followed, along with the experimental apparatus, form the most complete methodology, at this time, for the study of biofilms in porous media.

Jury :

  • Fabrice Golfier (Maître de conférence, Université de Lorraine - Laboratoire Géoressources) - Rapporteur
  • Laurent Oxarango (Professeur des Universités, Université Grenoble Alpes - Institut des Géosciences de l’Environnement) - Rapporteur
  • Etienne Paul (Professeur des Universités, INSA Toulouse - Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés) - Examinateur
  • Sabine Rolland du Roscoat (Maître de conférence, Université Grenoble Alpes - Laboratoire Sols, Solides, Structures, Risques) - Examinatrice
  • Pascal Swider (Professeur des Universités, Université de Toulouse - Institut de Mécanique des Fluides de Toulouse) - Directeur de thèse
  • Yohan Davit (Chargé de Recherche, CNRS - Institut de Mécanique des Fluides de Toulouse) - co-Directeur de thèse
  • Sophie Allart (Ingénieure de Recherche, INSERM - Centre de Physiopathologie de Toulouse Purpan) - Invitée
  • Michel Quintard (Directeur de Recherche, CNRS - Institut de Mécanique des Fluides de Toulouse) - Invité