The objective of this study was to examine excess biological phosphorus removing bacterial populations and their substrate utilization mechanisms. This study was a smaller part of a overall study of temperature effects upon excess biological phosphorus removal. Bacterial populations in both a continuous flow UCT (University of Cape Town) system and batch reactors were examined by direct counting using a well known staining procedure (Neisser staining), and a microscopic counting method developed by Cech and Hartman (1993). Substrate utilization was examined using PHB (Poly-β -Hydroxybutyrate - an internal substrate storage product) analysis by gas chromatography to supplement COD and acetate measurements. The results showed that Poly-P bacterial counts were significantly greater at a 5 day sludge age compared to a 10 day sludge age. It was noted from microscopic observations that the size of the poly-phosphate granules in the bacteria seemed to be a better indicator of system performance than the actual counts. It also was observed that the 'G' bacteria first described by Cech and Hartman (1993) were abundant at the 10 day sludge age but completely absent at the 5 day sludge age. PHB storage occurred in both the anaerobic zone and the first aerobic tank, and PHB utilization was seen in the subsequent aerobic tanks of the UCT system. The formation of PHB in the first aerobic reactor when no substrate was available supports the Mino (1987) model for excess biological phosphorus removal. In batch studies, substrate storage release were demonstrated in the aerobic zone. This explained why when acetate was present in the aerobic zone net phosphorus uptake didn't occur until all the acetate was utilized. When the temperature was lowered in the UCT system nitrification ceased. This resulted in soluble COD breakthrough into the aerobic zone, which stimulated filamentous growth, and eventually caused a lack of PHB formation. All of these factors contributed to a loss of excess biological phosphorus removal at the low temperature. / Master of Science