Lithoautotrophic nitrite-oxidizing bacteria (NOB) are known as fastidious microorganisms, which are hard to maintain and not many groups are trained to keep them in culture. They convert nitrite stoichiometrically to nitrate and growth is slow due to the poor energy balance. NOB are comprised of five genera, which are scattered among the phylogenetic tree. Because NOB proliferate in a broad range of environmental conditions (terrestrial, marine, acidic) and have diverse lifestyles (lithoautotrophic, mixotrophic, and heterotrophic), variation in media composition is necessary to match their individual growth requirements in the laboratory. From Nitrobacter and Nitrococcus relatively high cell amounts can be achieved by consumption of high nitrite concentrations, whereas accumulation of cells belonging to Nitrospira, Nitrospina, or the new candidate genus Nitrotoga needs prolonged feeding procedures. Isolation is possible for planktonic cells by dilution series or plating techniques, but gets complicated for strains with a tendency to develop microcolonies like Nitrospira. Physiological experiments including determination of the temperature or pH-optimum can be conducted with active laboratory cultures of NOB, but the attainment of reference values like cell protein content or cell numbers might be hard to realize due to the formation of flocs and the low cell density. Monitoring of laboratory enrichments is necessary especially if several species or genera coexist within the same culture and due to population shifts over time. Chemotaxonomy is a valuable method to identify and quantify NOB in biofilms and pure cultures alike, since fatty acid profiles reflect their phylogenetic heterogeneity. This chapter focusses on methods to enrich, isolate, and characterize NOB by various cultivation-based techniques.
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