Abstract:The nature and architecture of suspended matter (SM) and bacterial biofilms collected in Greater Nancy sewer system were investigated alongtwo years. Samples were taken from three sewer sections from upstream to downstream. Samples characterizations were conducted at millimetric scale using Confocal Laser Scanning Microscope (CLSM) and nanometric scale using Transmission Electron Microscopy (TEM). Several physicochemical and bacterial parameters were studied such as dissolved organic carbon (DOC), total bacteria, total suspended solid (TSS), volatile matter (VM) and particle size distribution of SM. The concentration of DOC decreases along the sewer from upstream to downstream. No clear evolution was found in total bacterial number along sewer. TSS was mainly composed of VM, and was found to decrease along the sewer. The temporal evolution of wastewater quality at a given sampling site shows no obvious change in total bacterial number. DOC and TSS concentrations were highest at midday, and decreases to reach their lowest value at 6h00. The volume size distribution of SM evolves from a multimodal distribution at upstream to a monomodal one at downstream of the sewer system. This decrease of particle size along sewer is likely related to the settling of SM and not to the degradation of suspended organic matter. Microscopic investigations indicated that SM and biofilm have similar composition (bacterial cells, extracellular polymeric substances (EPS), cellulose fibers, cell lyses detritus, organo-mineral detritus and mineral particles). Nevertheless, they have different 3D architectures. In SM, cellulose fibers form a skeleton for bacterial aggregates and biomass formation, within which bacteria may biodegrade fibers. The structural quantification revealed an open and patchy SM with 0.8 as porosity and 45% as coverage surface. Whereas, biofilms were more compact and denser than SM (porosity 0.55, coverage surface 92%). Water channels were identified through the biofilm depth with a decrease of areal porosity from the top to the bottom. Cellulose fibers were embedded in EPS matrix andbacterial division participates to biofilm growth. Viruses including bacteriophages were encountered within biofilms which may be considered as a potential reservoir of pathogenic viruses, especially during combined sewer overflows. Exchanges between SM and biofilms have been proven. On one handby the transfer of cellulose fibers from sewage to biofilms. On the other hand by the detachment of some parts from mature biofilms and their transport in sewage.