Relevance of Nitrospira for nitrite oxidation in a marine recirculation aquaculture system and physiological features of a Nitrospira marina-like isolate

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 C-13-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.