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Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides <i>Nitrospira</i>, "<i>Candidatus</i> Nitrotoga" has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of <i>Nitrotoga</i> in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent <i>Nitrotoga</i> sp. BS with regard to temperature and pH variations and evaluated its competitiveness against <i>Nitrospira defluvii</i> Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence <i>in situ</i> hybridization (FISH), and 16S amplicon sequencing, <i>Nitrotoga</i> sp. BS was clearly favored by its optimal growth parameters and dominated over <i>Ns. defluvii</i> at pH 7.4 and 17°C, whereas a pH of 6.4 was more selective for <i>Ns. defluvii</i> Our synthetic communities revealed that niche differentiation of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species.<b>IMPORTANCE</b> "<i>Ca.</i> Nitrotoga" is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between <i>Nitrotoga</i> and <i>Nitrospira</i> in bioreactors and identified conditions where the <i>K</i> strategist <i>Ns. defluvii</i> was almost replaced by <i>Nitrotoga</i> sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of <i>Nitrotoga</i> sp. BS versus <i>Ns. defluvii</i> at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel <i>Nitrotoga</i> cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.