5 — 1.5 1.32 × 10-4 1.64 × 10 -5 4.86 × 10-5 3.3 × 10 -5 9.48 × 10 -1 2.9 × 10 -5 6.29 × 10 -1 4.63 × 10 -6 1.5 — 3.0 1.2 × 10-5 1.98 × 10 -3 2.26 × 10-11 2.16 × 10 -3 1.22 × 10-5 1.78 × 10 -3 1.83 × 10-7 7.52 × 10 -1 Underlined text indicates statistically similar results, bold text indicates statistical increase and regular text indicates decrease. At DO = 0.5 mg R428 cell line O2/L, the transition from exponential phase to stationary phase resulted in a systematic decrease in relative mRNA concentrations of all four genes (Figure 3A4-C4 and Table 3). At DO

= 1.5 mg O2/L, this trend was valid for amoA, hao and norB. However, the stationary phase nirK relative mRNA concentrations were statistically higher than during exponential phase. At DO = 3.0 mg O2/L, only hao and norB displayed a decrease in relative mRNA concentrations upon transition from

exponential to stationary phase (Figure 3A4-C4, Table 3). In contrast, relative mRNA concentrations of amoA and nirK increased during stationary phase (Figure 3A4-C4, Table 3). Additionally, except at DO = 1.5 mg O2/L for nirK, the relative retention of amoA mRNA concentrations during stationary phase relative to exponential phase was consistently the highest (Figure 3 B4-C4). The retention factors averaged across all three DO concentrations were 1.98:1, 0.21:1, 1.86:1 and 0.08:1 for amoA, hao, nirK and norB, respectively (where a retention factor > 1) suggests relative increase during stationary AZD9291 phase). Table 3 Statistical comparison of relative mRNA concentrations and sOUR in exponential and stationary phase cultures grown at different DO concentrations (p ABT199 values < 5.0 × 10-2 indicate statistically significant differences). DO (mg O2/L) p =   amoA hao nirK norB sOUR 0.5 5.0 × 10-5 1.1 × 10-5 3.2

× 10-6 8.0 × 10-6 5.0 × 10 -1 1.5 5.5 × 10-6 6.4 × 10-8 7.7 × 10 -5 3.9 × 10-6 1.5 × 10-3 3.0 1.5 × 10 -3 6.3 × 10-4 5.1 × 10 -3 1.0 × 10-6 1.2 × 10 -1 Underlined text indicates statistically similar results, bold text indicates statistical increase and regular text indicates decrease. Impact of growth in the presence of added nitrite on N speciation, biokinetics and gene transcription Cell growth was not detected at an initial NO2 – concentration of 560 mg-N/L and DO = 1.5 mg O2/L, even after 2 weeks of incubation (data not shown). An initial NO2 – concentration of 280 mg NO2 –N/L and DO = 1.5 mg O2/L, resulted in a lag phase one day longer than that in the initial absence of nitrite (Figure 4 D1-D2 and Figure 2, B1-B2, respectively). However, the overall cell yield was not impacted. The extent of NH3 oxidized to NO2 – in the presence of 280 mg NO2 –N/L (88 ± 5%, n = 2) was not significantly different (α = 0.05) than in the absence of nitrite (90 ± 10%, n = 2). NH2OH accumulation was observed during the extended lag phase suggesting initial inhibition of NH2OH oxidation by NO2 – (Figure 4, D1).