One of the aims of wastewater treatment is the removal of phosphorus before the water is discharged into the environment. Since phosphorus concentrations in wastewater exceed the requirement of bacterial growth, biological phosphorus removal is based on the ability of a group of microorganisms, named “Polyphosphate Accumulating Organisms” (PAO), to store large quantities of intracellular phosphate in form of polyphosphate. In this study, we are focusing on the PAO actively involved in bioreactors operated with Aerobic Granular Sludge (AGS) technology. This process based on dense microbial biofilms is a cost-effective and landsaving alternative to the conventional biological wastewater treatment with activated sludge. This promising technology has received a significant commercial interest, but questions remain unanswered regarding the system performances in the context of full-scale applications. Like many natural microbial communities, the microbial structure of the AGS is complex. Its spatial organization is shaped by the diffusion of nutrients from the external environment and by the diffusion of microbial by-products formed in the biofilm. Understanding how the AGS microbial community works requires the elucidation of its spatial architecture and development. Light-sheet fluorescence microscopy is a powerful tool for examining microbial communities by overcoming the limitations of the classical three-dimensional confocal fluorescence microscopy. This technique shapes the excitation laser into a thin sheet providing an optical sectioning in order to illuminate the sample on a single plane. Then the emission signal is collected by a perpendicular lens. The large datasets generated are analyzed with a pipeline on the FIJI platform[1] to extract quantitative information. Preliminary results indicate that AGS are composed of heterogeneous biofilm aggregates. Interestingly, our observations are contrasting the AGS model structure previously described[2]. We would like to highlight the necessity of multiple observations when highly variable structures, like AGS, are analyzed in order to capture data which are representative of the studied system. [1] Schindelin et al. in Nat. Methods 28;9(7):676-82 (2012) [2] Winkler et al. in Water Res. 15;46(16):4973-80 (2012)   Poster 3 Conférence: Swiss Microbial Ecology Meeting 2019 (Lausanne) Titre: Anaerobic Biodegradation of organohalide pollutants: a crucial step towards the elucidation of proteins involved Auteurs: Lorenzo Cimmino, Adrian Schmid, Christof Holliger, Julien Maillard Abstract: Halogenated organic compounds (so-called organohalides) represent one of the major class of groundwater pollutants. The exploration of how organohalides are used as energy source is important in terms of ecosystem remediation but is also essential for the complete understanding of microbial metabolic interactions in the environment. Organohalide respiration (OHR) is a bacterial anaerobic process in which chlorinated compound, e.g. tetrachloroethene (PCE), is used as terminal electron acceptor. In the present work, Desulfitobacterium hafniense TCE1 and Dehalobacter restrictus, our model organohalide-respiring bacteria (OHRB) harbouring the pceABCT gene cluster, will be considered for the study of PCE respiration. To date, the function of PceA, the key catalytic enzyme in the process, and PceT, the dedicated molecular chaperone for PceA maturation, are well defined. However, the roles of PceB and PceC are not yet elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis of a possible PceABC respiratory complex is tempting but the question remains largely unanswered. The present work represents an evaluation of the stoichiometry of PceA, PceB and PceC proteins via quantitative proteomics applied to the membranes fractions of our model organisms. In a second phase, the use of Blue-Native electrophoresis technology will be considered to investigate whether PceC participates in a membrane-bound protein complex toge.