Online Monitoring of Aerobic Denitrification of Pseudomonas Aeruginosa by NAD(P)H Fluorescence
Abstract (Summary)In cystic fibrosis airway infection, Pseudomonas aeruginosa forms microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium’s metabolism at microaerobic conditions. Continuous cultures of P. aeruginosa (ATCC 9027) maintained at different dissolved oxygen concentrations (DO) and three different dilution rates (D) were studied for the effects of DO and D on various culture properties, especially on aerobic respiration and denitrification. The DO was varied from 0 mg/L (completely anoxic condition) to 2.2 mg/L, and measured with optical sensors that could accurately determine very low DO based on oxygen-quenched luminescence. The studied dilution rates were 0.026 h-1, 0.06 h-1 and 0.13 h-1. The strain was found to perform aerobic denitrification; while the specific nitrate and nitrite reduction rates decreased with increasing DO, denitrification persisted even at relatively high DO levels (1-2.2 mg/L) at different D. In the presence of nitrate, the Monod constant for DO (i.e., the critical DO at which the specific oxygen uptake rate (OUR) is half of the maximum rate) was practically zero (< 0.001 mg/L) for this P. aeruginosa strain. Aerobic denitrification appeared to function as an electron-accepting mechanism supplementary or competitive to aerobic respiration. The shift of culture’s respiratory mechanism was also clearly detected with a fluorometer targeting at intracellular NAD(P)H, i.e., the reduced coenzymes nicotinamide adenine dinucleotides (phosphate). Comparatively, the NAD(P)H fluorescence was highest at the anoxic, denitrifying condition (NFUDN), lowest at fully aerobic conditions (NFUOX), and intermediate fluorescence (NFU) at conditions where both denitrification and aerobic respiration occurred. Representing a quantitative measure of the culture’s “fractional approach” to the fully denitrifying state, the normalized fractions (NFU - NFUOX)/(NFUDN - NFUOX) were correlated with the calculated fractions of electrons accepted by denitrification. The denitrification-accepted fractions of electrons increased with the NFU fractions: the increases were gradual at larger DO levels (DO ? d 0.1 mg/L), but much sharper at lower DO at three different dilution rates. The fluorescence fraction changed more rapidly than the electron fraction at very low DO levels (< 0.001 mg/L). The results demonstrated that online NAD(P)H fluorescence was a feasible technique for effective monitoring and quantitative description of the microaerobic state of microorganisms.
School:The University of Akron
School Location:USA - Ohio
Source Type:Master's Thesis
Date of Publication:01/01/2006