Effect of environmental factors on denitrification gene expression and cumulative denitrification activity in Pseudomonas mandelii

Bacterial denitrification is an important biological process that contributes toward significant N2O accumulation into the atmosphere. The objective of this study was to quantify the effect of nitrate, pH and temperature on denitrification gene expression and cumulative denitrification activity in pure cultures of Pseudomonas mandelii. The effect of acetylene on denitrification gene expression in P. mandelii culture was also assessed. Denitrification gene expression in P. mandelii was quantified using quantitative reverse transcription real-time PCR. Denitrification activity was measured using the acetylene blockage method and gas chromatography to measure N2O accumulations in the headspace. P. mandelii cells in the presence or absence of nitrate, which acts as an electron acceptor in the denitrification process, were evaluated for cnorB gene expression and denitrification activity. After 4 h, only nitrate-amended cells maintained high levels of cnorB gene expression and demonstrated significant nitrous oxide accumulations. The impact of varying nitrate concentrations (0, 10, 100 and 1000 mg/L) on cnorB , nirS and nosZ gene expression was studied. Higher nitrate concentrations resulted in greater induction of nirS and cnorB gene expression and longer length of time that expression remained high. nosZ gene expression did not respond to NO3- treatment under the conditions tested. Acetylene was found to reduce nosZ gene expression, but did not affect nirS or cnorB gene expression. The effect of pH (5, 6, 7, 8) and temperature (10°C, 20°C, 30°C) on denitrification gene expression and activity were also investigated. Overall, cnorB and nirS gene expression was insensitive to pH values over the range of 6 to 8, but was substantially reduced at pH 5. Cumulative denitrification was highest in the pH 7 treatment, whereas negligible denitrification was measured at pH 5. Gene expression was sensitive to temperature with both the induction and time to achieve maximum gene expression being delayed as the temperature decreased from 30°C. Cumulative denitrification was lower for P. mandelii cells grown at 10°C and 20°C, compared to cells grown at 30°C. Lower pH and temperature negatively affected denitrification activity. This study demonstrated that environmental factors were able to impact denitrification at the level of gene expression and enzyme activity.