Characterization of a new heterotrophic nitrification bacterium Pseudomonas sp. strain JQ170 and functional identification of nap gene in nitrite production

Abstract

Heterotrophic nitrification bacteria play a critical role in nitrogen cycling and pollution removal. However, the underlying nitrification mechanisms are diverse and have rarely been investigated at the genetic level. In this study, the new heterotrophic nitrifier Pseudomonas sp. strain JQ170 was isolated. Strain JQ170 can utilize ammonia (NH4+-N), nitrite (NO2−-N), or nitrate (NO3−-N) as sole nitrogen sources, preferring NH4+-N. A ratio of 96.4% of 1.0 mM NH4+-N was removed in 24 h. The optimum pH, temperature, and carbon source for NH4+-N removal were pH 7.0, 30 °C, and citrate, at a C/N ratio of 9–18, respectively. During the NH4+-N removal process, only NO2−-N but neither hydroxylamine, NO3−-N, nor gaseous nitrogen were detected. A random transposon insertion mutagenesis library of strain JQ170 was constructed. Two NO2−-N-production deficient mutants were screened and transposon insertion sites were located in nap genes (which encode periplasmic NO3−-N reductase Nap). Further gene knockout and complementation of the napA gene confirmed nap as essential for NO2−-N production. The following nitrification processes in strain JQ170 is proposed: NH4+-N to NO3−-N in the cytoplasm; then NO3−-N to NO2−-N in the periplasmic space by Nap; finally, NO2−-N secreted out of cells. Overall, this paper provides new insight towards understanding heterotrophic nitrification at the genetic level.