Study of Nitrate Metabolism of Escherichia coli Using Fluorescence

Abstract

Public concern about environmental nitrate contamination has increased significantly. To investigate the nitrate metabolism of Escherichia coli, profiles of NAD(P)H fluorescence responding to nitrate/nitrite additions to anaerobic E. coli cultures 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 for the E. coli studied.

Aerobic Denitrification ofPseudomonas aeruginosaMonitored by Online NAD(P)H Fluorescence

“…Analyses of ammonium-N and NO x Ϫ -N (including both nitrate-N and nitrite-N) were made using an ammonia electrode (M-44325; Markson Science) (6,24). The ammonium concentrations could be measured accurately in a wide range (1 to 1,000 mg/liter of NH 4 ϩ -N), while the NO x Ϫ concentrations could be measured accurately only in the range of 1 to 20 mg/liter of NO x Ϫ -N. The sample was therefore diluted to the proper range prior to the analysis.…”

“…Universally present in living cells, the coenzymes NAD(P) are the major intermediate electron and hydrogen carriers, coupling substrate catabolism and respiration and anabolism (10,24). While NAD(P)H are fluorescent (excitation maximum of ϳ340 nm and fluorescence maximum of ϳ460 nm), their oxidized counterparts, NAD(P) ϩ , are not.…”

“…While NAD(P)H are fluorescent (excitation maximum of ϳ340 nm and fluorescence maximum of ϳ460 nm), their oxidized counterparts, NAD(P) ϩ , are not. The intensity of NAD(P)H fluorescence thus depends on the kinetic balance of their generation (by catabolism) and consumption (by respiration and anabolism) and is extremely sensitive to the change in cellular electron-accepting mechanisms (11,24,26).…”

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

“…Whole-cell fluorescence was used as a relative measure of reduced nucleotides in situ (43,45). Since only the reduced form NAD(P)H fluoresces at 460 nm, an immediate decrease in the fluorescence of fermenting cells was interpreted as a decline in the level of NAD(P)H and a decline in the NAD(P)H/NAD(P) ϩ ratio.…”

Rhamnolipid production byPseudomonas aeruginosa under denitrification: Effects of limiting nutrients and carbon substrates

“…Ammonium and nitrate analyses were made using an ammonia electrode (M-44325, Markson Science) (Clesceri et al, 1989;Trivedi and Ju, 1994). The ammonium concentrations could be measured accurately over a wide range (1 to 1000 mg/L of NH 4 + -N) whereas the nitrate concentrations were measurable over only 1 to 20 mg/L of NO 3 − -N. The sample was therefore diluted to the proper range prior to the analysis.…”