A bench-scale biological aerated filter (BAF) was operated under a range of substrate loadings and C/N influent ratios. Start-up with a substrate loading of 4.8 COD kg m−3 d−1 and an influent C/N ratio of 1.5 gave no nitrification, due to the high biofilm detachment rate caused by the daily backwashing. Subsequent runs with lower substrate loadings and backwashing rates allowed stable nitrification. A final run with the initial substrate loading and backwash rate gave stable nitrification. Oligonucleotide probes targeted to the 16S rRNA of the nitrifiers, as well as to other types of microorganisms, were used to characterize how the changes in loading and detachment affected the microbial structure in the biofilms. The proportion of nitrifiers decreased with increasing C/N ratio. The biofilm remaining after backwashing also contained a higher fraction of nitrifiers than did the backwash solids stripped from the outer layers of biofilm, which consisted mainly of active heterotrophs. Thus, stable nitrification was possible only when the nitrifiers became established deeper inside the biofilm.

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