Autotrophic microbial nitrification is the key process in the removal of ammonia from wastewater. To avoid the limitations of traditional microbiological methods an in situ identification technique for ammonia- and nitrite-oxidizing bacteria was developed. Based on comparative sequence analyses we designed a collection of 16S ribosomal RNA-targeted oligonucleotide probes for all validly described members of the genusNitrobacter . Whole cell hybridizations of target and reference cells with fluorescent probe derivatives were used to determine the optimal hybridization stringency for each of the probes. These probes were applied together with a recently developed probe for important members of the genus Nitrosomonas for simultaneous identification of ammonia- and nitrite-oxidizing bacteria in natural and engineered systems. Ammonia-oxidizing bacteria were identified in situ in river water, epiphytic biofilms from eutrophic wetlands, oligotrophic biofilms, a nitrifying trickling filter biofilm as well as in all analyzed nitrifying activated sludge samples. In none of these samples could Nitrobacter cells be detected in situ. However, all hitherto describedNitrobacter species and a strain of Nitrobacter sp. isolated from one of the analyzed nitrifying activated sludge samples showed bright hybridization signals with all Nitrobacter specific probes. Possible reasons for the absence of in situ detectable Nitrobacter cells are discussed.

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