The main goal of this study was to assess the impact that nitrate and light have on the relationship between the gene expression of the enzyme nitrate reductase and the incorporation of nitrate in the cosmopolitan diatom Thalassiosira pseudonana, both in laboratory experiments and in natural environments. Continuous cultures were grown at different nitrate (NO3-) concentrations (i.e., 60, 120, and 400 µM) to evaluate their effects on the expression levels of different genes of the nitrogen metabolic pathway (i.e., nitrate and ammonium transporters, nitrate and nitrite reductases, glutamine synthetases II and III). Semi-continuous cultures were grown under different irradiances (i.e., 50, 110, 200, and 320 µmol photon cm-2 s-1) to assess the influence of light intensity (irradiance) on the relationship between the expression of those genes, uptake, and assimilation of nitrate. The expression of all of the genes that were tested decreased significantly (p < 0.05) at the highest concentration of NO3- (i.e., 400 µM), with nitrate transporters showing the most pronounced change from 27.97 to 0.59 fold change cell-1 x 10-6, at 60 and 400 µM NO3- concentrations respectively. Ammonium transporters were detected at all concentrations of NO3-, suggesting that cells are always ready to metabolize ammonium. Growth was limited (µ = 0.99 d-1) by the low irradiance treatment, was maximum (µ = 2.04 d-1) at 200 µmol photon cm-2 s-1 and was inhibited (µ = 1.54 d-1) at the highest irradiance. These trends were reflected in gene expression and uptake rates, with minimum values at the lowest and highest irradiance levels. However, results from the enzymatic assay did not show any significant differences between treatments (p > 0.05). The trends observed in the enzymatic rates could be explained by the gene expression of NO3- reductase and the uptake and growth rates in a multiple regression analysis (R2 = 0.66, p < 0.05). The results of this study show that uptake is independent of gene expression, probably because of a decoupling between transcription and protein synthesis. Not all of the newly synthesized transcripts will inevitably be translated into proteins. And even if they were, there could be post-translational mechanisms preventing the enzymes to become active. This indicates that uptake can be independent of the expression. It was attempted to measure the expression of T. pseudonana genes involved in the metabolism of NO3- in natural diatom assemblages. The use of gene expression as a proxy for metabolic processes carried out by a phytoplankton assemblage in the field is limited and depends on environmental factors, since the current methods of assessing expression rely on genomic sequences that are particularly variable in phytoplankton. The assessment of gene expression provides a useful insight into physiological studies of phytoplankton, and it should be complemented with other measurements, such as the biomass and taxonomic composition of the assemblage for a more complete picture of marine ecosystem nutrient dynamics. / Graduate

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.