Strategies of Nitrosomonas europaea 19718 to counter low dissolved oxygen and high nitrite concentrations

Abstract

Background: Nitrosomonas europaea is a widely studied chemolithoautotrophic ammonia oxidizing bacterium. While significant work exists on the ammonia oxidation pathway of N. europaea, its responses to factors such as dissolved oxygen limitation or sufficiency or exposure to high nitrite concentrations, particularly at the functional gene transcription level are relatively sparse. The principal goal of this study was to investigate responses at the whole-cell activity and gene transcript levels in N. europaea 19718 batch cultures, which were cultivated at different dissolved oxygen and nitrite concentrations. Transcription of genes coding for principal metabolic pathways including ammonia oxidation (amoA), hydroxylamine oxidation (hao), nitrite reduction (nirK) and nitric oxide reduction (norB) were quantitatively measured during batch growth, at a range of DO concentrations (0.5, 1.5 and 3.0 mg O2/L). Measurements were also conducted during growth at 1.5 mg O2/L in the presence of 280 mg-N/L of externally added nitrite.

Results: Several wide ranging responses to DO limitation and nitrite toxicity were observed in N. europaea batch cultures. In contrast to our initial hypothesis, exponential phase mRNA concentrations of both amoA and hao increased with decreasing DO concentrations, suggesting a mechanism to metabolize ammonia and hydroxylamine more effectively under DO limitation. Batch growth in the presence of 280 mg nitrite-N/L resulted in elevated exponential phase nirK and norB mRNA concentrations, potentially to promote utilization of nitrite as an electron acceptor and to detoxify nitrite. This response was in keeping with our initial hypothesis and congruent with similar responses in heterotrophic denitrifying bacteria. Stationary phase responses were distinct from exponential phase responses in most cases, suggesting a strong impact of ammonia availability and metabolism on responses to DO limitation and nitrite toxicity. In general, whole-cell responses to DO limitation or nitrite toxicity, such as sOUR or nitrite reduction to nitric oxide (NO) did not parallel the corresponding mRNA (nirK) profiles, suggesting differences between the gene transcription and enzyme translation or activity levels.

Conclusions: The results of this study show that N. europaea possesses specific mechanisms to cope with growth under low DO concentrations and high nitrite concentrations. These mechanisms are additionally influenced by the physiological growth state of N. europaea cultures and are possibly geared to enable more efficient substrate utilization or nitrite detoxification.

Figure 1

Schematic of oxidative (unshaded enzymes) and reductive (gray shaded enzymes) nitrogen transformations in N. europaea (modified after [5]).

Figure 2

NH₃-N, NO₂^--N, and NH₂OH-N, (A1-C1), cell density and sOUR (A2-C2) profiles during N. europaea batch growth at DO = 0.5 mg/L (A), 1.5 mg/L (B) and 3 mg/L (C).

Figure 3

NO profiles and fraction of NO containing cells (A3-C3), and gene expression (A4-C4) during exponential phase and stationary phase at DO = 0.5 mg/L (A), 1.5 mg/L (B) and 3 mg/L (C) for cultures shown in Figure 2.

Figure 4

Profiles of NH₃-N, NO₂^--N, and NH₂OH-N (D1), cell density and sOUR (D2), NO and fraction of NO containing cells (D3) and gene expression (D4) during exponential phase and stationary phase at DO = 1.5 mg/L in the presence of added 280 mg NO₂^--N/L.