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In biofilters of recirculation aquaculture systems (RAS), nitrification by lithoautotrophic microorganisms is essential to prevent the cultivated organisms from intoxication with ammonium and nitrite. In moving-bed biofilters nitrifying microorganisms are immobilized together with heterotrophic bacteria in dense biofilms on carrier elements like plastic beads. Analyses of fatty acid profiles of these biofilms from a marine biofilter revealed a high abundance ofNitrospira-related lipid markers (8–12% of total fatty acids). Further results of a labeling experiment with 13C-bicarbonate in mineral salts medium with 3 mM nitrite confirmed thatNitrospirais the major autotrophic nitrite oxidizer in the biofilter system. According to 16S rRNA gene sequence analyses the nitrite-oxidizing community in the biofilter consisted of at least two different representatives ofNitrospira, one of which could be successfully isolated. The marine isolate ‘Ecomares 2.1’ belongs to cluster IVa and showed 98.8% 16S rRNA gene sequence similarity toNitrospira marina,whereas the enrichment ‘M1 marine’ is only distantly related (94.0% 16S rRNA gene sequence similarity toN. marina). In laboratory experiments, the isolate exhibited remarkable tolerances against high substrate and product concentrations (30 mM nitrite and 80 mM nitrate) as well as ammonium (50 mM). During the isolation process a strong tendency of this strain to develop biofilms became apparent. Thus, Ecomares 2.1 seems to be well adapted to the attached lifestyle in biofilters and the nitrogenous load prevailing in the effluent waters of RAS. Both members ofNitrospiracould be detected by PCR-based methods in environmental samples of marine and brackish RAS biofilters and are therefore considered to be characteristic for these engineered ecosystems.