Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Abstract

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.