Compound I of myeloperoxidase is capable of both one-electron oxidation and two-electron oxidation reactions. Halides and pseudohalides are the substrates for the two-electron oxidation and other compounds including a large variety of aromatic alcohols and amines can be oxidized via the single electron oxidation pathway. To investigate the catalytic mechanism of myeloperoxidase four structures of complexes of myeloperoxidase were solved. Two of them are complexes with hydroxamic acids and the other two are complexes with nitrite. Hydroxamic acids (salicylhydroxamic acid and benzylhydroxamic acid) can function as structural analogues for the aromatic alcohol and amine substrates of myeloperoxidase. The crystal structures of complexes of MPO with both hydroxamic acids have been solved at 1.85 Å resolution and their binding to myeloperoxidase is compared. The models show similar binding of their hydroxamic acid moieties but different orientations of their aromatic rings. The absence of the hydroxyl group covalently bound to the benzyl group in benzylhydroxamic acid creates an environment that does not permit the same favorable interactions with MPO when compared to salicylhydroxamic acid. These findings could explain the three orders of magnitude difference in the value of the dissociation constants of the two complexes. Nitrite has been shown to bind myeloperoxidase and also to reduce Compound I and Compound II. Crystal structures of the complex between myeloperoxidase and nitrite confirmed the binding of nitrite to the native enzyme both in the distal cavity and the chloride-binding site. The binding in the distal cavity occurred to the heme iron in the nitro mode. In the MPO-cyanide-nitrite ternary complex, nitrite had been shown to bind only at the chloride-binding site. No secondary site for nitrite binding had been seen in the distal cavity when cyanide was liganded to the iron. Overall, this study is the first to show from a crystallographic point of view a comparison in the mode of binding of the two hydroxamic acids to a mammalian peroxidase and also the binding of nitrite to a heme peroxidase.

The anaerobic oxidation of ammonium (anammox) with nitrite as electron acceptor is a microbial process that generates nitrogen gas as main final product. After being discovered in the Netherlands in the 1990s, anammox has been applied in state-of-the-art biotechnologies for the removal of N pollution from ammonium rich wastewaters. The anammox process offers significant advantages over traditional nitrification-denitrification based processes. Since anammox does not need elemental oxygen, it allows for important savings in aeration. Furthermore, due to the autotrophic nature of the bacteria, anammox does not require external addition of electron donor, often needed in systems with post-denitrification. Although the anammox bacteria have high specific activity, they are slow growing, with doubling times that can range from 10 to 25 d. Therefore, in case of a toxic event causing the death of the biomass, a long recovery period will be required to reestablish full treatment capacity. The purpose of this work is to investigate the inhibition of anammox bacteria by compounds commonly found in wastewaters, including substrates, intermediates and products of the anammox reaction. Among common wastewater constituents, sulfide was shown to be especially harmful, causing complete inhibition of anammox activity at concentrations as low as 11 mg H₂S L⁻¹. Dissolved oxygen was moderately toxic with a 50% inhibiting concentration of 2.3 and 3.8 mg L⁻¹ to granular and suspended anammox cultures, respectively. Among the various compounds involved in the anammox reaction, special attention was paid to nitrite. Numerous literature reports have indicated inhibition of anammox bacteria by its terminal electron acceptor. However to date, there is no consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition is impacted by variations in the physiological status of anammox cells. The mechanisms of anammox inhibition by nitrite were thoroughly investigated in batch and continuous experiments of this dissertation. The results of this work demonstrate that conditions hindering generation of metabolic energy have a detrimental effect on the tolerance of anammox cells to toxic levels of nitrite. The absence of ammonium during events of nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition, nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept that undissociated nitrous acid is the species responsible for the inhibition, the results in this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the free nitrous acid concentration. Nitrite stress triggered an active response of the anammox bacteria, which temporarily increased their ATP content to mitigate the inhibition. Additionally, starvation of anammox microorganisms, caused during storage or by sustained underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate exposure to nitrite. The results of this dissertation indicate that the tolerance of anammox bacteria to NO₂⁻ inhibition relies on limiting its accumulation in sensitive regions of the cell. Active metabolism in presence of NH₄⁺ allows for active consumption of NO₂⁻, avoiding accumulation of toxic intracellular NO₂⁻ concentrations. Furthermore, secondary active transport proteins may be used by anammox bacteria to translocate nitrite to non-sensitive compartments. Nitrite active transport relies on a proton motive force. Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which may disturb the maintenance of the intracellular proton gradient, will increase the sensitivity of anammox cells to NO₂⁻ inhibition. Strategies for the operation and control of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite under the absence of ammonium, low pH or after periods of starvation.

Este trabalho descreve o desenvolvimento de um método eletroanalítico para determinação sequencial de nitrito (NO2-) e nitrato (NO3-), utilizando como técnica, a voltametria de pulso diferencial. O método se baseia na redução eletroquímica dos íons nitrato sobre um ultramicroeletrodo de ouro modificado in situ com cádmio depositado em regime de subtensão, e na seqüência, a remoção da monocamada de cádmio e a oxidação eletroquímica dos íons nitritos sobre o ultramicroeletrodo não modificado. Os ensaios voltamétricos para determinação quantitativa de nitrato e nitrito foram realizados em solução de NaClO4 0,1 molL-1 + HClO4 1,0x 10-3 molL-1 (pH = 3,3) preparada com água ultrapura. Utilizando as condições experimentais e os parâmetros voltamétricos otimizados, foram construídas curvas analíticas para determinação de nitrito e nitrato separadamente e também para determinação sequencial dos dois analitos. Para a determinação do NO2-, foi observado uma relação linear entre a corrente de pico e a concentração desse íon dentro do intervalo de concentração de 1,0 x 10-5 molL-1 a 1,1 x 10-4 molL-1, com um limite de detecção igual a 1,151 ± 0,091 µmolL-1 e limite de quantificação igual a 3,838 ± 0,091 µmolL-1. Para a determinação do NO3-, também foi observado uma relação linear entre corrente de pico e concentração desse analito dentro do intervalo estudado, que foi de 2,00 x 10-5 molL-1 a 2,50 x 10-4 molL-1. O limite de detecção encontrado foi 4,839 ± 0,275 µmolL-1 e o limite de quantificação 16,131 ± 0,275 µmolL-1. A determinação sequencial de nitrito e nitrato foi avaliada dentro do intervalo de concentração de 5,00 x 10-5 molL-1 a 2,50 x 10-4 molL-1 para NO3- e 1,00 x 10-5 molL-1 a 4,50 x 10-5 para NO2-. Para ambos os casos, a relação entre corrente de pico versus concentração do analito foi linear. Para a determinação sequencial os limites de detecção são 16,177 ± 0,794 µmolL-1 para NO3- e 2,243 ± 0,179 µmolL-1 para NO2- e os limites de quantificação são 53,922 ± 0,794 µmolL-1 para o NO3- e 7,476 ± 0,179 µmolL-1 para o NO2-. Os limites de detecção, os limites de quantificação e demais parâmetros estatísticos apresentados nesse trabalho, foram obtidos a partir de cálculos baseados em procedimentos descritos em Miller e Miller68 e Silva69. / This work describes the development of an electroanalytical method for sequential determination of nitrite (NO2-) and nitrate (NO3-), using as a technique, differential pulse voltammetry. The method is based on the electrochemical reduction of nitrate ions on a gold ultramicroelectrode modified in situ by underpotential deposition of cadmium, and subsequently, the removal of cadmium monolayer and the electrochemical oxidation of nitrite on ultramicroelectrode unmodified. The voltammetric analysis for quantitative determination of nitrate and nitrite were carried out in NaClO4 0.1 molL-1 + HClO4 1.0 x 10-3 molL-1 (pH = 3.3) prepared with ultrapure water. Using the optimized experimental conditions and voltammetric parameters, analytical curves were constructed for determination of nitrite and nitrate separately and for sequential determination of the two analytes. The relationship between peak current and concentration of NO2- were found to be linear in the concentration range between 1.0 x 10-5 molL-1 and 1.1 x 10-4 molL-1, with a detection limit of 1.151 ± 0.091 µmolL-1 and quantification limit of 3.838 ± 0.091 µmolL-1. For determination of NO3- was also observed a linear relationship between peak current and concentration of analyte within the concentration range studied, which was from 2.00 x 10-5 molL-1 to 2.50 x 10-4 molL-1. The detection limit was 4.839 ± 0.275 µmolL-1 and the quantification limit was 16.131 ± 0.275 µmolL-1. The sequential determination of nitrite and nitrate was assessed within concentration range from 5.00 x 10-5 molL-1 to 2.50 x 10-4 molL-1 for NO3- and from 1.00 x 10-5 molL-1 to 4.50 x 10-5 for NO2-. In both cases, the relationship between peak current versus analyte concentration were found to be linear. The detection limits for sequential determination are 16.177 ± 0.794 µmolL-1 for NO3- and 2.243 ± 0.179 µmolL-1 for NO2- and the quantification limits are 53.922 ± 0.794 µmolL-1 for NO3- and 7.476 ± 0.179 µmolL-1 for NO2-. The detection and quantification limits and other statistical parameters presented in this work were obtained from calculations based on procedures described in Miller and Miller68 and Silva69.

Losses of soil nitrogen that cannot be attributed to leaching or crop removal have been observed in many field experiments. Several mechanisms have been proposed to account for these losses. Perhaps the best known mechanism involves the process of microbial denitrification. Undoubtedly this process plays a major role in nitrogen loss but it does not seem to adequately account for many of the losses observed. A thorough understanding of other possible loss pathways has tremendous agricultural implications as well as being of interest from a purely scientific standpoint. It has long been accepted that nitrite is an intermediate in the biological oxidation of ammonium to nitrate. Because of the high reactivity of nitrous acid and the nitrite ion many investigators have proposed pathways involving them. Considering the diversity of possible reactions and products involving nitrite it is not surprising that numerous contradictions are found in the literature on this subject. The work reported here was an attempt to clarify the role of some of these pathways in the destruction of the nitrite ion in acid soils. The availability of a gas chromatograph and incubation equipment made the study feasible from a technical standpoint. Most of the data reported here were collected by the author in the late summer of 1961, using techniques developed over the previous year. Some data re included that were collected by Dr. Keith Justice in the summer of 1962 using these same methods.

Supplemental carbon has been successfully added and implemented at biological nutrient removal treatment plants all around the world in order to reach low nitrogen discharge limits. Although, methanol has been the most prevalent external electron donor used due to its low cost and effectiveness, many utilities are moving away from it due to cost volatility, safety issues, and hindered performance in cold weather conditions. Many sustainable and alternative sources are being researched, such as glycerin-based products (Rohrbacher et al., 2009), sugar-based waste products (Pretorius et al., 2007), and effluents from food and beverage industries (Swinarski et al., 2009). Four 22-L sequencing batch reactors (SBRs) were utilized to investigate four different supplemental carbon sources: 100% reagent grade methanol, 100% reagent grade glycerol, bio-diesel glycerol waste, and an industrial waste alcohol. These reactors were operated at 20�"C with a 15 day solids retention time. Intensive profiles were carried out three times a week to monitor performance and collect data to calculate COD consumption: nitrate-nitrogen denitrified (C: N) ratios. The glycerol and bio-diesel glycerol waste reactors performed similarly as they both exhibited significant and consistent nitrite accumulation during the entire experiment. Based on reactor restart, nitrite accumulation was evident and significant within two days after startup and consistent for all further operation. Rapid nitrate to nitrite reduction coincident with COD uptake was also observed. The two glycerol reactors demonstrated an increased carbon demand over time. The commonly reported hypothesis that activated sludge transitions from a generalist population of ordinary heterotrophic organisms (OHO) that use substrate, glycerol in this case, less efficiently, producing low yields and slow growth rates, to a specialist population that use glycerol more efficiently, with higher yields and slightly faster growth rates, was verified. This is known as the generalist-specialist theory. While this hypothesis appears to be supported from an overall analysis of the data, the actual mechanism seems to be intracellular glycerol storage coincident with rapid nitrate to nitrite denitrification, followed by slow nitrite reduction to nitrogen gas. This can possibly lead to degradation of the internally stored glycerol in the aerobic zones of the following cycle, implying a significant economic impact with glycerin addition. Although this has not been investigated further, it is believed that the presence of glycogen-accumulating organisms (GAOs) could be responsible for this intracellular storage of glycerol resulting in partial denitrification and accumulation of nitrite. The methanol and waste alcohol reactors also performed similarly to each other and neither of these reactors exhibited any nitrite accumulation upon carbon addition. The specific denitrification rate (SDNR) of the waste alcohol was slightly higher and increased more rapidly than for the methanol reactor. The C: N for these two reactors was comparable, and methanol was close to the expected value of 4.8 g COD utilized/ g nitrate-N denitrified. The C: N for the waste alcohol during steady state operation was somewhat higher than expected. The waste alcohol exhibited an �"alcoholic�" odor upon addition to the reactors during startup, but this issue diminished as the biomass became acclimated to the waste alcohol. Both industrial waste alcohol and glycerol can be considered viable alternatives to methanol; however, glycerol supplementation for denitrification can be problematic. If the glycerol dose is not optimized, then partial denitrification is observed and will lead to nitrite in the effluent, causing an increased chlorine demand for plants applying chlorine for disinfection. This is thought to occur due to energy limitations resulting from carbon storage and thus, using glycerol at treatment plants performing biological phosphorus removal (BPR) or enhanced biological phosphorus removal (EBPR) might see inefficient removal due to selective carbon utilization by polyphosphate-accumulating organisms (PAOs), or due to competition between PAOs and GAOs. Although denitrification of nitrate to nitrite occurs more quickly with prolonged glycerol addition, it also results in an increased carbon demand which causes a significant impact economically. / Master of Science

<p> Nitrate assimilation is the process by which nitrate is converted into ammonia, and ultimately into organic nitrogenous compounds, which are then made available to organisms which require an exogenous supply of organic nitrogen. Nitrite is an intermediate in this process and the mechanism of its conversion to ammonia, which is catalyzed by the enzyme nitrite reductase, needs clarification. </p> <p> The purpose of this investigation was to find a suitable assay system for nitrite reductase in N. crassa and to examine some aspects of the metabolic control of the enzyme. A new assay system for nitrite reductase is described and evidence suggesting that the enzyme is derepressible is presented. </p> / Thesis / Master of Science (MSc)

<p> Nitrite is an intermediate in the pathway of nitrate assimilation. Several questions about reduction beyond the level of nitrite remain to be answered. </p> <p> The purpose of this investigation was to induce mutants deficient in nitrite reduction and to characterize these mutants phenotypically and genetically in an attempt to answer the following questions: </p> <p> (a) How many enzymes are required in vivo for nitrite reduction? </p> <p> (b) How many genes control nitrite reduction? </p> <p> (c) Is nitrite reductase localized within a particle? </p> <p> The results of this investigation showed that nitrite reductase is controlled by at least three genes and three cistrons on two linkage groups. None of the 'nitrite-mutants' were allelic with nitrate reductase mutants. There appeared to be more than one type of nitrite reductase activity in extracts of repressed wild type mycelia. Only one of these nitrite reductase species seemed to be necessary for the reduction of nitrite in vivo. </p> / Thesis / Master of Science (MSc)

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