Although enhanced biological phosphorus removal (EBPR) and complete biological nutrient removal (BNR) systems can be operated successfully by experienced operators, the accuracy of design and strength of the scientific background need to be reinforced to enable accurate modeling and economically optimal design. One way to accomplish this would be through a better understanding of the biochemical mechanisms and microbial population dynamics that determine the reliability and efficiency of EBPR, and the utilization of this information to improve the design and operation of BNR plants. Such knowledge will also contribute to better structure of modeling tools that are used for design and educational purposes. The current body of knowledge is limited to observational studies that lack detailed biochemical explanations backed with a series of well planned experiments, and this has introduced uncertainties and inaccuracies into the biochemical and design models. Therefore, this study mainly covers a biochemical survey of the underlying metabolisms of active populations in BNR sludges. BNR biomass with biological phosphorus removal (BPR) capability was cultivated in continuous flow reactor (CFR) systems, configured as either University of Cape Town (UCT) and anoxic/oxic (A/O) systems. Following an acclimation period at 20°C, low temperature stress (5°C) was imposed on one UCT system for investigation of the response of the microbial consortium responsible from EBPR activity under cold temperature. Once a stable population with EBPR capabilities is established in each system, activities of ten enzymes that are hypothesized to be taking part in the EBPR metabolism were measured. These enzymes were selected among those that take part in major known pathways of bacterial energy and growth metabolism. Also, 13C-NMR was used as a tool to monitor the flux of labeled carbon in and out of pools of cellular storage; i.e. glycogen and polyhydroxyalkanoates (PHA). Combining the gathered information, accurate mass balances of carbons and reducing equivalents were calculated, eventually leading to determination of the biochemical pathways utilized by the EBPR consortium. Additionally, anaerobic stabilization of COD, a long debated but empirically established phenomenon, was addressed during the study. Considering the pathways proposed to be operative under different conditions imposed on the EBPR systems, a biochemical explanation for the occurrence of COD stabilization in wastewater treatment systems that incorporate anaerobic zones was proposed. Accordingly, depending on the pathways actively used by a microbial consortium, electrons stored in NADH and FADH2 can either be transferred to the terminal electron acceptor, oxygen, or they can be incorporated into storage polymers such as glycogen for future use. Such differences in metabolism reflect in the quantity of the oxygen consumed in the aerobic reactors. Thus, the correct incorporation of anaerobic stabilization of COD into process design would reduce design aeration requirements and result in economic savings during both construction and operation. / Ph. D.

Synaptic long-term modifications following neuronal activation are believed to be at the origin of learning and long-term memory. Recent experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states of a single synapse. The biochemical network involving calcium/calmodulin-dependent protein kinase II (CaMKII) and its regulating protein signaling cascade has been hypothesized to durably maintain the synaptic state in form of a bistable switch. Furthermore, it has been shown experimentally that CaMKII and associated proteins such as protein kinase A and calcineurin are necessary for the induction of long-lasting increases (long-term potentiation, LTP) and/or long-lasting decreases (long-term depression, LTD) of synaptic efficacy. However, the biochemical mechanisms by which experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such networks are still unknown. We present a detailed biochemical model of the calcium/calmodulin-dependent autophosphorylation of CaMKII and the protein signaling cascade governing the dephosphorylation of CaMKII. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentrations. Repetitive high calcium levels switch the system from a weakly- to a highly phosphorylated state (LTP). We show that the reverse transition (LTD) can be mediated by elevated phosphatase activity at intermediate calcium levels. It is shown that the CaMKII kinase-phosphatase system can qualitatively reproduce plasticity results in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. A reduced model based on the CaMKII system is used to elucidate which parameters control the synaptic plasticity outcomes in response to STDP protocols, and in particular how the plasticity results depend on the differential activation of phosphatase and kinase pathways and the level of noise in the calcium transients. Our results show that the protein network including CaMKII can account for (i) induction - through LTP/LTD-like transitions - and (ii) storage - due to its bistability - of synaptic changes. The model allows to link biochemical properties of the synapse with phenomenological 'learning rules' used by theoreticians in neural network studies.

Synaptic long-term modifications following neuronal activation are believed to be at the origin of learning and long-term memory. Recent experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states of a single synapse. The biochemical network involving calcium/calmodulin-dependent protein kinase II (CaMKII) and its regulating protein signaling cascade has been hypothesized to durably maintain the synaptic state in form of a bistable switch. Furthermore, it has been shown experimentally that CaMKII and associated proteins such as protein kinase A and calcineurin are necessary for the induction of long-lasting increases (long-term potentiation, LTP) and/or long-lasting decreases (long-term depression, LTD) of synaptic efficacy. However, the biochemical mechanisms by which experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such networks are still unknown. We present a detailed biochemical model of the calcium/calmodulin-dependent autophosphorylation of CaMKII and the protein signaling cascade governing the dephosphorylation of CaMKII. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentrations. Repetitive high calcium levels switch the system from a weakly- to a highly phosphorylated state (LTP). We show that the reverse transition (LTD) can be mediated by elevated phosphatase activity at intermediate calcium levels. It is shown that the CaMKII kinase-phosphatase system can qualitatively reproduce plasticity results in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. A reduced model based on the CaMKII system is used to elucidate which parameters control the synaptic plasticity outcomes in response to STDP protocols, and in particular how the plasticity results depend on the differential activation of phosphatase and kinase pathways and the level of noise in the calcium transients. Our results show that the protein network including CaMKII can account for (i) induction - through LTP/LTD-like transitions - and (ii) storage - due to its bistability - of synaptic changes. The model allows to link biochemical properties of the synapse with phenomenological 'learning rules' used by theoreticians in neural network studies.