Dynamic changes in spatial microbial distribution in mixed-population biofilms were experimentally determined using a microslicer technique and simulated by a biofilm accumulation model (BAM). Experimental results were compared with the model simulation. The biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. Experimental results showed that an increase of substrate loading rate (i.e., organic carbon and NH4-N) resulted in the microbial stratification in the biofilms. Heterotrophs defeated nitrifiers and dominated in the outer biofilm, whereas nitrifiers were diluted out in the outer biofilm and forced into the inner biofilm. At higher organic loading rates, a stronger stratified microbial spatial distribution was observed, which imposed a severe internal oxygen diffusion limitation on nitrifiers and resulted in the deterioration of nitrification efficiency. Model simulations described a general trend of the stratified biofilm structure. However, the actual stratification was stronger than the simulated results. For implication in the reactor design, when the specific carbon loading rate exceeds a certain limit, nitrification will be deteriorated or require a long start-up period due to the interspecies competition resulting in oxygen diffusion limitation. The extend of microbial stratification in the biofilm is especially important for determination of feasibility of nitrification in the presence of organic matters.

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