Nitrite-oxidizing bacteria (NOB) are responsible for the second step of nitrification in natural and engineered ecosystems. The recently discovered genus Nitrotoga belongs to the Betaproteobacteria and potentially has high environmental importance. Although environmental clones affiliated with Nitrotoga are widely distributed, the limited number of cultivated Nitrotoga spp. results in a poor understanding of their ecophysiological features. In this study, we successfully enriched the nonmarine cold-adapted Nitrotoga sp. strain AM1 from coastal sand in an eelgrass zone and investigated its physiological characteristics. Multistep-enrichment approaches led to an increase in the abundance of AM1 to approximately 80% of the total bacterial population. AM1 was the only detectable NOB in the bacterial community. The 16S rRNA gene sequence of AM1 was 99.6% identical to that of "Candidatus Nitrotoga arctica," which was enriched from permafrost-affected soil. The highest nitrogen oxidation rate of AM1 was observed at 16°C. The half-saturation constant (K_m ) and the generation time were determined to be 25 μM NO₂^- and 54 h, respectively. The nitrite oxidation rate of AM1 was stimulated at concentrations of <30 mM NH₄Cl but completely inhibited at 50 mM NH₄Cl. AM1 can grow well under specific environmental conditions, such as low temperature and in the presence of a relatively high concentration of free ammonia. These results help improve our comprehension of the functional importance of NitrotogaIMPORTANCE Nitrite-oxidizing bacteria (NOB) are key players in the second step of nitrification, which is an important process of the nitrogen cycle. Recent studies have suggested that the organisms of the novel NOB genus Nitrotoga were widely distributed and played a functional role in natural and engineered ecosystems. However, only a few Nitrotoga enrichments have been obtained, and little is known about their ecology and physiology. In this study, we successfully enriched a Nitrotoga sp. from sand in a shallow coastal marine ecosystem and undertook a physiological characterization. The laboratory experiments showed that the Nitrotoga enrichment culture could adapt not only to low temperature but also to relatively high concentrations of free ammonia. The determination of as-yet-unknown unique characteristics of Nitrotoga contributes to the improvement of our insights into the microbiology of nitrification.

Kentaro Soh

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