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 of Nitrospira-related lipid markers (8-12% of total fatty acids). Further results of a labeling experiment with (13) C-bicarbonate in mineral salts medium with 3 mM nitrite confirmed that Nitrospira is 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 of Nitrospira, 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 to Nitrospira marina, whereas the enrichment 'M1 marine' is only distantly related (94.0% 16S rRNA gene sequence similarity to N. 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 of Nitrospira could 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.

Biofi lters are crucial and costly components in marine recirculating aquaculture systems. However, not much is known about the settlement of nitrifying organisms and developing nitrifi cation rates during the start-up phases of these reactors. The nitrifying microorganisms in moving bed biofi lters of two marine recirculation aquaculture systems identical in construction were monitored for 388 and 477 days by PCR based methods, accompanied by laboratory nitrifying activity tests. Ammonia and nitrite were added to the recirculating aquaculture system 1, while system 2 was spiked with fi sh feed. On day 88, system 1 was stocked with turbot (at 17 °C) and on day 126 system 2 was stocked with sea bass cultivated at 22 °C. The potential nitrifi cation rates corresponded well to the conditions in the operating systems, and in both systems slowly developing nitrite oxidation rates led to high nitrite peaks. However, after 218 (biofi lter 1) and 286 (biofi lter 2) days, potential rates of nitrite oxidizing bacteria outreached those of ammonia oxidizing bacteria.The nitrite oxidizing bacteria were remarkably diverse on the genus level, and for the fi rst time Nitrotoga was detected in marine biofi lter systems. Nitrospira was assumed to be the most dominant nitrite oxidizing bacterium, also confi rmed by electron microscopy. The ammonia oxidizing organisms belong almost exclusively to Nitrosomonas, of which dominant species shifted in both systems over time. The high similarities of some 16S rRNA gene sequences of Nitrospira and Nitrosomonas to sequences found previously in other marine recirculating aquaculture systems suggested that the species are characteristic for this artifi cial ecosystem.

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