The population distribution of nitrifying and heterotrophic bacteria in biofilm particles from BAS reactors has been studied with the aid of an oxygen microelectrode. The oxygen fluxes to the biofilm surface calculated from the external diffusion profile corresponded well with volumetric uptake measurements in batch tests using a large number of particles. Measurements of internal oxygen concentrations in these small biofilm particles (radius: ≈ 0.5 mm) proved to be reproducible. The internal structure of the biofilm appeared to be highly heterogeneous, i.e. oxygen profiles differed between different sites on a biofilm particle and between different particles from the same sample. Despite the lateral heterogeneity it was possible to obtain a clear indication for the existence of a layered structure of nitrifying and heterotrophic bacteria in these biofilms. Simulation of the biofilm formation by a standard biofilm simulation model also (BIOSIM) revealed this layered structured. The segregation of autotrophs and nitrifiers was, however, to be stronger than could be described by BIOSIM. The results underline that for obtaining a stable conversion of substrates by slow growing organisms (ammonium, xenobiotics) a not too high biofilm surface specific substrate loading of fast degradable COD is required to ensure sufficient oxygen penetration to the layer with the slow-growing microorganisms.

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