Orientadores: Theo Guenter Kieckbusch, Koichi Fujie / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Quimica / Made available in DSpace on 2018-08-04T03:36:42Z (GMT). No. of bitstreams: 1 Haraguchi_LilianHiromi_M.pdf: 2787215 bytes, checksum: b71f02a7f8c97e582d66c8c4ca3c8bf8 (MD5) Previous issue date: 2005 / Resumo: O controle do lodo em excesso, proveniente dos processos de tratamento de águas residuais industrial e doméstica é, cada vez mais, um assunto de considerável preocupação, pois o descarte destes resíduos pode causar sérios danos ao meio ambiente. Em vista disto, o desenvolvimento de novas tecnologias que permitam uma diminuição na quantidade de lodo produzido torna-se indispensável. Um outro problema encontrado no tratamento de águas residuais é o descarte de alguns nutrientes provenientes do processo, como o fosfato, em cursos d¿água, o que tem aumentado a eutrofização em rios. Um processo chamado Remoção Biológica Aprimorada de Fosfato ¿ EBPR (Enhanced Biological Phosphate Removal), é, atualmente, considerado um dos meios mais econômicos para remoção de fosfato. O presente trabalho teve como objetivo investigar a viabilidade técnica de tratar o lodo em excesso em condições sub e supercríticas da água e reutiliza-lo como fonte de carbono biodegradável no processo EBPR. Lodo em excesso de uma indústria de processamento de peixes (Toyohasshi ¿ Japão) foi submetido às reações hidrotérmicas em um reator em batelada, a uma faixa de temperatura de 200 '400 GRAUS¿C, pressões variando de 1,8 a 30 Mpa e tempo de reação de 10 min. Ensaios de liberação de fosfato foram conduzidos com o objetivo de analisar a capacidade dos microorganismos em liberar fosfato intracelular em meio anaeróbio, utilizando o lodo tratado como fonte de carbono... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: the control of excess sludge produced in the industrial and domestic wastewater treatment plants has been a matter of considerable concern. Discharge of excess sludge poses significant risks to the environment and the development of new technologies that are able to suppress the pollution have challenged many researches. On the other hand, the release of some nutrients like phosphorus in wastewater streams increases the eutrophication problem in many rivers throughout the world with the consequent growth of algae induced by high levels of phosphate. Enhanced Biological Phosphate Removal (EBPR) process has been currently considered one of the most economical ways to remove phosphate from the wastewaters. The objective of this work is to investigate the feasibility of reusing excess sludge from a fish processing industry located in Japan was treated by hydrothermal reactions, at reaction temperature ranging from 200 to '400 DEGREES¿, pressure of 1,8 to 30 MPa and fixed reaction time of 10 min. Experiments on phosphorus release under anaerobic conditions were also carried out using the treated excess sludge as carbon source. For the hydrothermal conditions tested, the results showed that the solubilization and the biodegradability were improved as evidenced by its content change after hydrothermal reactions... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.

Waste Stabilisation Ponds (WSPs) are characterised by low phosphorus (P) removal capacity. Heterotrophic bacteria are principal microbial agents in WSPs in addition to algae. As treatment proceeds in WSPs, algal growth increases and pH rises, this has lead to believe that P removal is mainly through sedimentation as organic P algal biomass and precipitation as inorganic P. In activated sludge treatment plants (AS), microbial P removal has been improved and is termed as enhanced biological phosphorus removal. There was a need to establish whether it was possible to enhance P removal in WSPs. A performance assessment of pond system at the University of Dare s Salaam (UDSM), Tanzania, has shown that 90% of the P removed was in the primary pond (facultative) and the rest in the maturation pond (aerobic). In these studies, a pure strain A. hydrophyla was isolated from an activated sludge wastewater treatment plant in Sweden. This plant has a train that functions with enhanced biological phosphorus removal. The strain was tested for P uptake in minimal media supplemented with glucose, succinate or acetate, grown aerobically and anaerobically/aerobically. This strain was able to take up P without having been subjected to the anaerobic phase. It was observed that P uptake was enhanced after the anaerobic phase with media supplemented with glucose, but not with succinate or acetate. Phosphorus uptake repeatedly followed the bacterial growth pattern with correlation coefficients of more than 95%. Therefore P removal has a direct correlation with bacterial growth. Two isolates Acinetobacter sp. (isolated from the primary facultative pond) and E .coli (isolated from the maturation pond) were obtained from a tropical WSP treatment system at the UDSM. They were subjected to aerobic P uptake experiment similar to those of A.hydrophyla. The uptake per unit absorbance of bacterial growth was found to be comparable to that of A.hydrophyla, isolated from AS. These results showed that heterotrophic activity is important in WSPs. It is possible to enhance P removal in these systems by designing the primary ponds for maximum heterotrophic activity and probably enrichment. / QC 20101119

It is generally accepted that a decrease in temperature causes the rates of chemical and biochemical reactions to slow down, usually resulting in poorer performance of biological wastewater treatment systems. Despite this, early researchers repeatedly showed that excess biological phosphorus removal (EBPR) was more efficient at colder temperatures. Recent studies, however, have demonstrated that the reaction rates of EBPR processes decrease with temperature in accordance with Arrhenius' Law, resulting in an apparent contradiction in the literature. The objective of this study was to investigate the EBPR temperature controversy. The experimental systems used were two, lab-scale UCT configuration plants fed with acetate as the sole volatile fatty acid (VFA) source. The results showed that EBPR systems do perform more efficiently at colder temperatures, i.e., at 5°C compared to 20°C. The reason for better system performance was determined to be related to reduced competition for substrate in the non-oxic zones that results in an increased population of phosphate accumulating organisms (PAOs) relative to non-PAOs and, therefore, greater EBPR efficiency even though the reaction rates are slower. The proliferation of PAOs relative to non-PAOs at cold temperature indicates that some of the PAOs are psychrophilic, i.e., they have alternate biochemical pathways that give them a competitive advantage over bacteria dependent upon glycogen metabolism. The activated sludge acclimated to 20°C had relatively high polyhydroxyvalerate (PHV) and glycogen contents relative to sludge acclimated to 5°C. It was initially hypothized that there is a significant competition between PAO and glycogen accumulating organisms (GAOs) at 20°C and cold temperature (5°C) nearly eliminates this competition in favor of the PAOs. A series of batch test experiments revealed that despite similar acetate utilization by the sludges grown at the two temperatures nearly 30% less PHA was produced by the sludge taken from the 20°C reactor, indicating that GAOs were a small fraction of the population at 20°C. Transmission electron microscopy pictures showed that the biomass acclimated to 20°C had a much more diverse bacterial population than the biomass acclimated to 5°C. However, no GAO population was detected in electron microscopy samples under any temperature conditions. The decreased P removal efficiency at 20°C was then attributed to the presence of fermentative or other non poly-P bacteria that are capable of utilizing substrate under anaerobic conditions. PHA production greatly increased at 5°C, whereas glycogen metabolism substantially reduced. Even though glycogen is an essential requirement for EBPR mechanism, the EBPR microorganisms have the ability to adapt their metabolic pathways to environmental conditions and greatly reduce their need for glycogen. It is apperant that cold temperature inhibits some of the key enzymes in glycogen metabolism resulting in lower glycogen accumulation that in turn increases the EBPR performance. Therefore temperature not only exerts selective pressure on the dominant population but also alters the metabolic pathways of the EBPR process. Increased glycogen accumulation, as observed in this study at 20°C, may not be related to GAO proliferation as suggested by Filipe et al. (2001) instead it may be related to EBPR bacteria to efficiently use glycogen metabolism. Current models (Brdjanovic et al. 1997; Filipe et al. 2002) consider that GAO metabolism is an integral part of EBPR metabolism and the performance of EBPR processes depends on PAO/GAO fraction in the EBPR system. No GAO proliferation was observed even the A/O process was operated without P addition for more than 3 weeks at 10°C. Therefore such important concept should be further investigated before it is included in EBPR models. EBPR stoichiometry was presumed to be insensitive to temperatures (Brdjanovic et al. 1997). However, observed stoichiometric values of PHA storage per unit glycogen utilization and PHA utilization per unit glycogen rephlenishment were quite different at different temperatures. Temperature, therefore, not only affects the kinetics of EBPR systems but also affects the EBPR stoichiometry. Most prokaryotic cells have the ability to alter their cellular membrane fatty acid composition as temperature decreases to counteract the adverse effects of temperature on membrane fluidity (Becker et al., 1996). This unique ability is known as "homeoviscous adaptation". In this study, homeoviscous adaptation by EBPR activated sludge was investigated for a series of temperatures ranging from 20°C to 5°C using one of the lab scale EBPR systems. The fatty acid analysis results showed that the unsaturated to saturated fatty acid ratio increased from 1.40 to 3.61 as temperature dropped from 20 to 5°C. The increased cis-9-hexadecanoic acid (C16:1) at 5°C strongly indicated the presence of homeoviscous adaptation in the EBPR bacterial community. Thus the cell membranes of the EBPR community were still in a fluid state, and solute transport and proton motive force mechanisms were operable even at 5°C. It was concluded that loss of EBPR performance at low temperatures, as reported by McClintock et al. (1992) was not related to the physical state of the cellular membranes, but was probably caused by unsuitable operational conditions. Even though the transport of volatile fatty acids (e.g. acetate) is an integral part of EBPR biochemistry and stoichiometry, this important concept has been ignored. Fleet (1997) concluded that acetate entry into bacterial cells in EBPR sludge was simple passive diffusion based upon the results of a single study (Baronofsky et al. 1984). However, this study showed that neither acetate nor propionate can cross the cell membrane via simple passive diffusion. The existence of apparent saturation curves when the substrate uptake rates (acetate and propionate) were plotted against the substrate concentrations suggested that transport of volatile fatty acids obey facilitated or active transport. Following from the above results, an investigation of the impacts of operational conditions such as low solids retention time (SRT), presence of electron acceptors in the non-oxic zones, low anaerobic detention time, and lack of acclimation was performed. The results showed that the "critical, i.e., wash-out" SRT increased as temperature decreased, but if the biomass was permitted to acclimate to the lower temperature, a major population shift would occur which would increase the capacity of the system for phosphorus (P) removal. When the 5 °C sludge was allowed to acclimate at a relatively high SRT (18 d), the system's P-removal capacity greatly surpassed that of the 20 °C system. The decrease in EBPR performance because of the presence of nitrates in the non-oxic zones was determined to be greater than what would be predicted based on accepted stoichiometry. / Ph. D.

Thesis (Master of Environmental Management)--Cape Peninsula University of Technology, 2018. / Fresh water is a renewable resource, but it is also finite, especially given environmental impacts from anthropogenic activities. Globally, there are countless signs that untreated industrial discharge into fresh watercourses is one of the main causes of ecosystem degradation. Poultry slaughterhouse wastewater (PSW) amongst the main pollutants of fresh water sources. In recent years, the world’s pre-eminent researchers have developed innovative wastewater treatment processes to treat the large quantity of wastewater generated as well as to manage the environmental health concerns arising from PSW discharged into the environment. Furthermore, increasing wastewater treatment capital costs and the implementation of increasingly rigorous government legislation to mitigate environmental pollution whilst minimizing fresh water source contamination, requires that wastewater such as PSW, be adequately treated prior to discharge. In order to assist the small-scale poultry producers in South Africa (SA), process simulation for a small-scale poultry slaughterhouse wastewater treatment plant was proposed using Sumo Wastewater treatment plant (WWTP) simulation software. Sumo is an innovative and most versatile wastewater simulation package on the market. The simulator is capable of modelling treatment plants of unlimited complexity, focusing largely on Biochemical oxygen demand (BOD), Chemical oxygen demand (COD), nitrogen and phosphorus removal; with digester, and side streams design options, being available. Considering the possible advantages in modelling and ongoing studies of implementing wastewater treatment to increase water management, anaerobic digestion of high strength wastewater such as PSW, warranted this research study. Model development from the simulation included the evaluation of numerous design options to assist small scale poultry producers, to have a variety of designs to choose from in their PSW WWTP designs. With the aid of Sumo, two models were designed in this study, namely a single-stage and a two-stage anaerobic digestion without a recycle. The PSW used as feed was obtained from a local poultry slaughterhouse (Western Cape, South Africa). Both model designs predicted the reduction of the organic matter (COD, BOD5) total suspended solids (TSS), and volatile suspended solids (VSS) in the PSW. The digester for the single stage anaerobic digestion system modelled was set to operate at steady state for 150 days under mesophilic temperature (35 ˚C) with a solid retention time (SRT) of 25 days. The COD, TSS, VSS and BOD removal efficiencies reached a maximum of 64%, 77%, 84%, and 94%, respectively, at an organic load rate (OLR) of 143.6 mg COD/L/day. A minute increase in the ammonia (NH3) and phosphate (PO3- 4) concentration was observed once the simulation was completed. As for the two-stage anaerobic digestion system, both digesters were set to perform at mesophilic temperatures (35 ˚C) and a SRT of 13 days in the first digester and 25 days in the subsequent digester. The two-stage anaerobic digestion showed better performance in comparison to the single-stage anaerobic digestion system. The COD, TSS, VSS and BOD5 removal efficiencies reached a maximum of 69%, 79%, 85%, and 96%, respectively, at an at an OLR of 143.6 mg COD/L/day. A similar trend regarding phosphate and ammonia removal was noticed in the two-stage anaerobic digestion, suggesting a tertiary treatment system to be in place for further treatment. Although, the two-stage anaerobic digestion demonstrated adequate performance, for the purpose of this study, the single-stage was the process recommended for PSW treatment, as it is less costly and will be suitable for small scale poultry producers; albeit biogas production is much higher when digesters are connected in series. The PSW treatment modelling for this study was successfully employed with the resultant effluent being compliant with the City of Cape Town (CCT) wastewater and industrial effluent by-law discharge limits. Although, both the PO3- 4 and NH3 were suggested to require further monitoring. Therefore, the poultry slaughterhouse from which the PSW was obtained will be able to safely discharge the treated wastewater proposed in this research into local water bodies, i.e. rivers in the Western Cape, SA; however, the treated PSW will not be suitable for re-use as process water.

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Aktivt slam är en biologisk process som används i stor utsträckning för rening av avloppsvatten, där bakterier som odlas under luftiga förhållanden avlägsnar organiska ämnen och näringsämnen från förorenat vatten. För industriell verksamhet modelleras denna process för att representera och förutsäga biologiska fenomen. De modeller som används för närvarande (främst modellen för aktiverat slam) ger acceptabla resultat men har vissa begränsningar, t.ex. lång kalibreringstid och beroende av många modellparametrar som är svåra att förutsäga. Under detta projekt som genomfördes på företaget SUEZ bedömdes det om sådana modeller kunde förbättras genom att lägga till ett termokemiskt perspektiv i deras arkitektur, antingen genom att minska antalet parametrar eller genom att teoretiskt förutsäga deras värden. Det termokemiska bidraget bedömdes på två olika nivåer: de stökiometriska förhållanden som beskriver systemet och de biologiska processernas kinetik. På grundval av en omfattande litteraturstudie valdes två metoder ut för att föra in termodynamiska överväganden i dessa delar av modellen. Termodynamiska beräkningar som krävs för att förbättra specifikationen av de förorenande molekyler som finns i vattenmatrisen. Arkitekturen i den ursprungliga modellen för aktiverat slam ändrades för att möjliggöra en differentiering mellan substrat av olika slag. När det gäller den stökiometriska studien visade det sig att den dynamiska förutsättningen av termodynamiska begränsningar för reaktionerna inte var av något större intresse med tanke på de stora mängder energi som finns tillgängliga. Användningen av olika organiska substrat gav dock intressanta resultat, eftersom det gjorde det möjligt att modellera de biologiska beteendena på ett mer detaljerat sätt och att förutsäga tillväxten av specifika typer av mikroorganismer. Den kinetiska studien gav måttliga resultat, med adekvata simuleringar men utan någon ny större förbättring av modellen. Den mest lovande utvecklingen av denna studie tycks vara en modell som omfattar en mängd olika organiska substrat med motsvarande biomassapopulationer, för att kunna rikta in sig på den specifika förekomsten av intressanta bakterier. / Activated sludge is a biological process that is widely used for wastewater treatment, where bacteria grown in aerated conditions remove organic substances and nutrients from polluted waters. For industrial operations, this process is modeled to represent and predict biological phenomena. Currently used models (mainly, the Activated Sludge Model) give acceptable results but suffer from some limitations such as long calibration time and dependency on an many model parameters that are difficult to predict. During this project conducted at the company SUEZ, it was assessed whether such models could be improved by adding a thermochemical perspective to their architecture, either by reducing the number of parameters involved or by predicting theoretically their values. The contribution of thermochemistry was assessed on two different levels: the stoichiometric relations that describe the system, and the kinetics of the biological processes. Based on an extensive literature study, two methods were selected to inject thermodynamical considerations to these parts of the model. Thermodynamic calculations required to improve the specification of the pollutant molecules present in the water matrix. The architecture of the initial Activated Sludge Model was modified to allow for a differentiation between substrates of different nature. Regarding the stoichiometric study, it appeared that the dynamic prevision of thermodynamical constraints over the reactions did not show a major interest considering the high amounts of energy available. However, the use of different organic substrates gave interesting results because it allowed to model more finely the biological behaviors and to predict the growth of specific types of microorganisms. The kinetic study gave mitigated results, with adequate simulations but no new major improvement to the model. The most promising development of this study appears to be a model involving a multiplicity of organic substrates, with corresponding biomass populations, to target the specific occurrence of bacteria of interest.

Activated sludge treatment systems rely on a microbial consortium structurally organized in bioflocs to treat pollutants present in wastewater. The treatment process efficiency in these systems can be severely affected by toxic chemicals present in the influent wastewater. The effects of chemical toxins at the treatment process level are determined by the mechanisms that occur at the biofloc and cellular levels, which can be physical, chemical and physiological in nature. We believe that the overall process effects of chemical toxins on activated sludge systems likely result from a combination of all three types of mechanisms and that they are interdependent, in the sense that specific bacterial stress response mechanisms (physiological mechanisms that protect the cell from toxic conditions) may lead to physical/chemical alterations at the floc level, and vice-versa. Ultimately, understanding the mechanisms that occur at the floc and metabolic scales will help to design more robust and efficient treatment systems, and to develop tools to prevent and mitigate the effects of toxic chemicals on activated sludge systems. In this research, we set out to establish the link between the effects of chemical toxins on activated sludge cultures at the process, floc and metabolic scales. First, the effects of shock loads of different toxic sources (1-chloro-2,4-dinitrobenzene (CDNB), cadmium, 1-octanol, 2,4-dinitrophenol (DNP), weakly complexed cyanide, pH 5, 9 and 11, and high ammonia levels) on activated sludge process parameters (biomass growth, respiration rate, flocculation, chemical oxygen demand (COD) removal, dewaterability and settleability) were studied. For all chemical shocks except ammonia and pH, concentrations that caused 15, 25 and 50% respiration inhibition were used to provide a single pulse chemical shock to sequencing batch reactor (SBR) systems containing a nitrifying (10 day solids retention time – SRT) and a non-nitrifying (2 day SRT) biomass. We found that cadmium and pH 11 shocks were the conditions that most detrimentally affected all the processes, followed by CDNB. DNP and cyanide primarily led to effects on respiration, while pH 5, 9, octanol and various ammonia concentrations did not impact the treatment process to a significant extent. Additionally, there was a clear correlation between biomass deflocculation and increases in the effluent soluble COD of the shocked reactors for different chemical sources. With this study, we were able to establish a source-effect matrix linking classes of chemical toxins to their potential inhibitory effects on activated sludge processes, thereby contributing to a better understanding of the potential effects of toxic industrial discharges into biological treatment systems. The findings of the first phase of the research, specifically the correlation between chemical-induced deflocculation and increases in soluble COD, served as a motivation to explore the role of floc structure in the response of activated sludge cultures to toxic compounds, and to conduct a more in-depth analysis of the supernatant (soluble phase) of toxin-exposed activated sludge. In one study, we evaluated the respiration inhibition induced by octanol, cadmium, N-ethylmaleimide (NEM), cyanide and DNP on activated sludge biomasses with different floc structures but similar physiological characteristics, with the objective of assessing the role of the extracellular polymeric substances (EPS) in flocs as a protection barrier against chemical toxins. Mechanical shearing was applied to fresh mixed liquor to produce biomasses with different floc structure properties and specific oxygen uptake rate assays were conducted on the sheared and unsheared mixed liquors. The results showed that the respiration inhibition by octanol and cadmium was more intense in sheared mixed liquor (which had less EPS material available in the flocs and smaller floc sizes) than in the unsheared biomass. Conversely, the respiration inhibition induced by NEM and cyanide was similar for the different mixed liquors tested. These results allowed us to conclude that the EPS matrix functions as a protective barrier for the bacteria inside activated sludge flocs to chemicals that it has the potential to interact with, such as hydrophobic (octanol) and positively-charged (cadmium) compounds, but that the toxicity response for soluble, hydrophilic toxins (NEM and cyanide) is not significantly influenced by the presence of the polymer matrix. In the final study that was conducted, we used the metabolomics-based technique metabolic footprinting to assess if the soluble phase of mixed liquor exposed to different chemical toxins exhibited a toxin-specific biochemical composition. We hypothesized that toxin-specific effects could be distinguished through footprint patterns of those soluble samples. The impact of cadmium, DNP and NEM shock loads on the composition of the soluble fraction of activated sludge mixed liquor was analyzed by liquid chromatography-mass spectrometry (LC-MS). The results from this study indicated that there was a significant release of biomolecules (proteins, carbohydrates and humic acids) from the floc structure into the bulk liquid due to chemical stress. More importantly, using a multivariate statistical method called discriminant function analysis with genetic algorithm variable selection (GA-DFA), we were able to show that the soluble phase samples from the different reactors could be differentiated, thereby indicating that the footprints generated by LC-MS were different for the four conditions tested and, therefore, toxin-specific. These footprints, thus, contain information about specific biomolecular differences between the samples, and we found that only a limited number of m/z (mass to charge) ratios from the mass spectra data was needed to differentiate between the control and each chemical toxin-derived samples. In addition, since the experiments were conducted with mixed liquor from four distinct wastewater treatment plants, the discriminating m/z ratios may potentially be used as universal stress biomarkers. These results are promising and indicate that LC-MS may be used for the discovery of activated sludge stress biomarkers, to allow the development of new toxin detection technologies for prevention of upset events in activated sludge systems. / Ph. D.

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.