A number of innovative nitrogen removal technologies have been developed to address the treatment challenges caused by stringent regulations and increasing chemical and energy cost. A major contributing factor to these challenges is the liquid stream originating from the process of dewatering anaerobically digested solids. This liquid, also knows as centrate, reject water or sludge liquor, can cause an increase of up to 25% in ammonia loading. The recently discovered anaerobic ammonia oxidation (anammox) process is a major breakthrough for treatment of these streams as it has the potential to remove up to 85% of nitrogen load without external carbon source addition. The anammox process is combined with another process that oxidizes half of the ammonia to nitrite (nitritation) in a separate reactor such as in the SHARON process, or in the same reactor such as in the DEaMmONification (DEMON) process. Despite intensive laboratory research for the last 10 years to fully understand these processes, there is still a high level of skepticism surrounding the implementation of full-scale systems. The reason for this skepticism could be due to frequent failures observed in the lab scale systems as well as reported slow bacterial growth. We think that this technology might be used more effectively in the future if process kinetics, inhibition and toxicity can be better understood. This work focused on the DEMON process with a goal to understand the kinetics and inhibition of the system as a whole and the anammox process in particular. A DEMON pilot study was undertaken at the Alexandria Sanitation Authority (ASA) and had several study participants, including ASA, the District of Columbia Water and Sewer Authority (DCWASA), CH2M Hill Inc., Envirosim Ltd, the University of Innsbruck and Virginia Tech. We investigated the growth rate of anammox bacteria within a quasi-optimal environment. Laboratory-scale experiments were conducted to assess anaerobic ammonia oxidation inhibition by nitrite as well as aerobic ammonia oxidation inhibition by compounds present in the DEMON reactor feed, such as a defoaming agent, a sludge conditioning polymer, and residual iron from phosphorus removal practices. The study revealed that the DEMON process can be efficiently controlled to limit nitrite accumulation capable of causing process inhibition. The target ammonium loading rate of 0.5 kg/m3/d was reached, and no upset was noticed for a loading up to 0.80 kg/m3/d with an HRT of 1.7 days. The ammonia removal efficiency reached an average of 76% while total nitrogen removal efficiency had an average of 52%. Most of the process upsets were caused by aerobic ammonia oxidation failure rather than anammox inhibition. Failure in ammonia oxidation affected pH control, a variable which is at the center of the DEMON process control logic. The pilot study is summarized in Chapter 3 of this Dissertation. The low anammox maximum specific growth rate (µmax,An) as well as nitrite inhibition are historically reported to be the major process challenges according to the literature, but the degree to which each contributes to process problems differs widely in the literature. In this study, we estimated µmax,An by using the high F:M protocol commonly used for nitrifying populations. We also studied the effect of both short term and sustained nitrite exposure on anammox activity. In this study, µmax,An was estimated to be 0.017 h-1. The study results also suggest that anammox bacteria can tolerate a spike of nitrite-N at concentrations as high as 400 mg/L as long as this concentration is not sustained. Sustained concentrations above 50 mg/L caused a gradual loss of activity over the long term. Finally, the inhibition of aerobic ammonia oxidizing bacteria (AerAOB) observed in the DEMON reactor was investigated using laboratory experiments and is reported in Chapter 6. AerAOB inhibition was, in most cases, the main reason for process upset. Compounds that were suspected to be the cause of the inhibition were tested. The study noticed that a defoaming agent, polymer and ferrous iron had some inhibiting properties at the concentrations tested. / Ph. D.

Este trabalho iniciou-se com o objetivo de superexpressar proteínas com atividade ATPase, como tentativa de alterar a conservação de energia livre na levedura S. cerevisiae, de maneira a aumentar o rendimento da fermentação alcoólica. Para isso, duas ATPases nativas de S. cerevisiae, as chaperonas codificadas pelos genes HSP104 e CDC48, foram superexpressas, individualmente, sob o controle de quatro promotores de diferentes forças, provocando diferentes gastos energéticos na levedura. Entretanto, não foi possível obter aumento no rendimento em etanol. Em seguida, foi feito um estudo que visou comparar essas linhagens em situação de estresse térmico, ácido ou osmótico, tipicamente encontrados no processo brasileiro de produção de etanol. A 40 °C, uma linhagem superexpressando CDC48 apresentou velocidade específica máxima de crescimento 17 % maior que a linhagem de referência, indicando maior tolerância ao estresse térmico. Finalmente, avaliou-se Hsp104 e Cdc48 em um contexto fisiológico no qual as atividades dessas proteínas pudessem ser mais requeridas. Como as chaperonas moleculares são conhecidas por agirem como primeira linha de defesa contra a formação de proteínas incorretamente enoveladas e agregados proteicos, estudaram-se a morfologia e a fisiologia da superexpressão de HSP104 e CDC48 em linhagens com desarranjo no controle de qualidade de proteínas intracelulares, causado por mutações no proteassomo 20S. A superexpressão de CDC48 ou HSP104 reverteu em parte a morfologia alterada de alguns desses mutantes de proteassomo. / The initial goal of this work was to overexpress proteins with ATPase activity in Saccharomyces cerevisiae, as an attempt to alter the conservation of free energy in this yeast, in order to increase alcoholic fermentation yield. Therefore, two native S. cerevisiae ATPases, the chaperones encoded by HSP104 and CDC48, were individually overexpressed under the control of four promoters with different strengths, in order to provoke different levels of energy expenditure. Increments in the ethanol yield could not be observed in any of the constructed strains. Subsequently, a study was carried out to compare these mutant strains with reference strains under heat, acid or osmotic stress, which are typically found in the industrial fuel ethanol production in Brazil. At 40 oC a strain overexpressing CDC48 displayed a maximum specific growth rate 17 % higher than that of the reference strain, indicating a greater tolerance to heat stress. Finally, Hsp104 and Cdc48 were evaluated in a physiological context in which the activity of these proteins would be required in a higher level. Since molecular chaperones are known to act as the first defense line against the formation of misfolded proteins and aggregates, the physiological and morphological effects of HSP104 or CDC48 overexpression were analyzed in strains with protein quality control disarrangements caused by mutations in proteasome 20S. The overexpression of either CDC48 or HSP104 partially reversed the altered morphology of some of these proteasome mutants.

Este trabalho iniciou-se com o objetivo de superexpressar proteínas com atividade ATPase, como tentativa de alterar a conservação de energia livre na levedura S. cerevisiae, de maneira a aumentar o rendimento da fermentação alcoólica. Para isso, duas ATPases nativas de S. cerevisiae, as chaperonas codificadas pelos genes HSP104 e CDC48, foram superexpressas, individualmente, sob o controle de quatro promotores de diferentes forças, provocando diferentes gastos energéticos na levedura. Entretanto, não foi possível obter aumento no rendimento em etanol. Em seguida, foi feito um estudo que visou comparar essas linhagens em situação de estresse térmico, ácido ou osmótico, tipicamente encontrados no processo brasileiro de produção de etanol. A 40 °C, uma linhagem superexpressando CDC48 apresentou velocidade específica máxima de crescimento 17 % maior que a linhagem de referência, indicando maior tolerância ao estresse térmico. Finalmente, avaliou-se Hsp104 e Cdc48 em um contexto fisiológico no qual as atividades dessas proteínas pudessem ser mais requeridas. Como as chaperonas moleculares são conhecidas por agirem como primeira linha de defesa contra a formação de proteínas incorretamente enoveladas e agregados proteicos, estudaram-se a morfologia e a fisiologia da superexpressão de HSP104 e CDC48 em linhagens com desarranjo no controle de qualidade de proteínas intracelulares, causado por mutações no proteassomo 20S. A superexpressão de CDC48 ou HSP104 reverteu em parte a morfologia alterada de alguns desses mutantes de proteassomo. / The initial goal of this work was to overexpress proteins with ATPase activity in Saccharomyces cerevisiae, as an attempt to alter the conservation of free energy in this yeast, in order to increase alcoholic fermentation yield. Therefore, two native S. cerevisiae ATPases, the chaperones encoded by HSP104 and CDC48, were individually overexpressed under the control of four promoters with different strengths, in order to provoke different levels of energy expenditure. Increments in the ethanol yield could not be observed in any of the constructed strains. Subsequently, a study was carried out to compare these mutant strains with reference strains under heat, acid or osmotic stress, which are typically found in the industrial fuel ethanol production in Brazil. At 40 oC a strain overexpressing CDC48 displayed a maximum specific growth rate 17 % higher than that of the reference strain, indicating a greater tolerance to heat stress. Finally, Hsp104 and Cdc48 were evaluated in a physiological context in which the activity of these proteins would be required in a higher level. Since molecular chaperones are known to act as the first defense line against the formation of misfolded proteins and aggregates, the physiological and morphological effects of HSP104 or CDC48 overexpression were analyzed in strains with protein quality control disarrangements caused by mutations in proteasome 20S. The overexpression of either CDC48 or HSP104 partially reversed the altered morphology of some of these proteasome mutants.