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

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Title: Effect of environmental factors on denitrification gene expression and cumulative denitrification activity in Pseudomonas mandelii
Author: Saleh-Lakha, Saleema
Department: Department of Environmental Biology
Advisor: Trevors, Jack T.Goyer, Claudia
Abstract: 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.
URI: https://hdl.handle.net/10214/24980
Date: 2009
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