Microelectrodes for O2 and NO2/NO3 and fluorescently labelled 16S rRNA-targeted oligonucleotide probes were combined to examine the activity and stratification of nitrifying bacteria in a trickling filter biofilm. Microprofiles showed that O2 consumption and NO3/NO2 production were restricted to the upper 50-100 μm of the biofilm. The vertical distribution of the nitrifying bacteria Nitrosomonas sp. and Nitrobacter sp. was investigated by fluorescent in situ hybridisation (FISH) with specific oligonucleotides. Nitrifiers formed a dense layer of cells and cell clusters in the upper part of the biofilm. This correlates well with the measured activity profiles.

Ammonia- and nitrite-oxidisers occurred in close vicinity to each other supporting a fast sequential metabolism from ammonia to nitrate. Both species were not restricted to the oxic part of the biofilm, but also appeared -in lower numbers- in the anoxic layers on the bottom of the biofilm.

A short term decrease in the O2 concentration of the bulk water resulted in a quick decrease in O2 penetration and metabolic rates inside the biofilm. However, neither the stratification nor the cellular ribosome content of nitrifiers changed within a few hours.

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