AbstractPublic concern about environmental nitrate contamination has increased significantly. To investigate the nitrate metabolism ofEscherichia coli, profiles of NAD(P)H fluorescence responding to nitrate/nitrite additions to anaerobicE. colicultures at the resting or minimum‐growth state were monitored by an on‐line fluorometer. Two different types of fluorescence profiles were observed. Upon nitrate addition, the fluorescence signal dropped instantaneously, indicating the presence of NAD(P)H‐dependent nitrate/nitrite reductases, without previous nitrate/nitrite induction. The signal remained at that level until all of the nitrate was reduced. In some cases, the signal then directly rose back to the earlier anaerobic level. In others, it partially recovered to an intermediate level, remained at that level for a period much longer than the first stage, and then returned to the anaerobic level. The single‐stage response implied that the nitrite formed by nitrate reduction was totally converted to ammonium simultaneously. The two‐stage response, however, resulted from intermediate nitrite accumulation: the rise in fluorescence at the end of the first stage signified nitrate depletion, and the second stage corresponded to reduction of the nitrite accumulated in the first stage. This is supported by the specific rates of nitrate and nitrite reduction determined from the fluorescence profiles. While the former (0.0138 ± 0.0026 g of NO3−‐N/(g of cells)‐h) was about the same in all the runs conducted, the latter was found to be 1 order of magnitude lower than the former in cultures showing the two‐stage response. The presence of ammonium, at 0–50 ppm NH4+‐N, was found to have no appreciable effect on the nitrate reduction rate
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