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Nitrification is a significant concern for drinking water systems employing chloramines for secondary disinfection. Utilities have implemented a range of disinfection strategies that have varying levels of effectiveness in the prevention and control of nitrification events, including optimizing the chlorine-to-ammonia ratio, maintaining chloramine residual throughout the distribution system, controlling pH, and temporal switching to free chlorination. Annual or semi-annual application of free chlorination is practiced by 23% of chloraminating systems on a temporary basis as a preventative measure, even though it has the undesirable consequences of temporarily increasing disinfection byproducts, facilitating coliform detachment, and altering water taste and odor. Although temporal free chlorination and other nitrification control methods have been widely studied in the field and in pilot-scale systems, very little is known about the stress responses of nitrifying bacteria to different disinfection strategies and the role physiological state plays in the resistance to disinfection. It is well known that many commonly studied bacteria, such as Escherichia coli, are able to better resist disinfection by free chlorine and chloramines under nutrient limitation through regulation of stress response genes that encode for DNA protection and enzymes that mediate reactive oxygen species. We compared the genomes of E. coli and the ammonia-oxidizing bacterium Nitrosomonas europaea, and found that many of the known stress response mechanisms and genes present in E. coli are absent in N. europaea or not controlled by the same mechanisms specific to bacterial growth state. These genetic differences present a general susceptibility of N. europaea to disinfection by chlorine compounds. Using an experimental approach, we tested the hypothesis that N. europaea does not develop increased resistance to free chlorine and monochloramine during starvation to the same degree as E. coli. In addition, N. europaea cells were challenged with sequential treatments of monochloramine and hypochlorous acid to mimic the disinfectant switch employed by drinking water utilities. Indicators of activity (specific nitrite generation rate) and viability (LIVE/DEAD® BacLight© membrane-integrity based assay) were measured to determine short-term effectiveness of disinfection and recovery of cells over a twelve day monitoring period. The results of disinfectant challenge experiments reinforce the hypothesis, indicating that the response of N. europaea to either disinfectant does not significantly change during the transition from exponential phase to stationary phase. Exponentially growing N. europaea cells showed greater susceptibility to hypochlorous acid and monochloramine than stationary phase E. coli cells, but had increased resistance compared with exponential phase E. coli cells. Following incubation with monochloramine, N. europaea showed increased sensitivity to subsequent treatment with hypochlorous acid. Complete loss of ammonia-oxidation activity was observed in cells immediately following treatment with hypochlorous acid, monochloramine, or a combination of both disinfectants. Replenishing ammonia and nutrients did not invoke recovery of cells, as detected in activity measurements during the twelve day monitoring period. The results provide evidence for the effectiveness of both free chlorine and chloramines in the inhibition of growth and ammonia-oxidation activity in N. europaea. Furthermore, comparison of viability and activity measurements suggest that the membrane integrity-based stain does not serve as a good indicator of activity. These insights into the responses of pure culture nitrifying bacteria to free chlorine and monochloramine could prove useful in designing disinfection strategies effective in the control of nitrification. / Master of Science

This item was digitized as part of the Million Books Project led by Carnegie Mellon University and supported by grants from the National Science Foundation (NSF). Cornell University coordinated the participation of land-grant and agricultural libraries in providing historical agricultural information for the digitization project; the University of Arizona Libraries, the College of Agriculture and Life Sciences, and the Office of Arid Lands Studies collaborated in the selection and provision of material for the digitization project.

A greenhouse experiment was conducted to study the effectiveness of Dwell, a nitrification inhibitor, on the efficiency of two fertilizers applied to three Arizona soils, Anthony sandy loam, Laveen loam, and Vinton loamy sand. Tomato plants Lycopersicum esculentum cultivar Row Pak were used as an index plant. These plants were grown in soil columns of 10 cm internal diameter and 50 cm in length, containing 4,500 g of soil. Two fertilizers, urea and ammonium sulfate (AS), were applied at two rates: 112 and 224 kg N/ha. The nitrification inhibitor Dwell was applied also at two rates: 0 and 1.27 kg/ha⁻¹ of active ingredient. The active ingredient in the chemical is 5- ethoxy -3- (trichloromethyl)-1, 2,4- thiadizole. Both fertilizers and Dwell were added to the soils using two methods: (1) uniform mixtures, in which the fertilizer and Dwell were mixed with the top 500 cm³ soil volume in the columns, and (2) banded fertilizers and Dwell, in which a soil paste, made by mixing 10 ml of 0.02% Dwell solution with the volume of 50 cm³ soil, was added to the required amount of the fertilizer to soil column about 7cm below the soil surface. This factorial designed experiment was arranged in a randomized complete block with three replications of each treatment. Plants were allowed to grow for 67 days after transplanting to the soil columns. At the end of the experiment, samples from two depths (0 to 18 cm and 18 5o 40 cm) were analyzed for NH₄, NO₃, and organic N. Dry matter yield and N content of plants were also determined. Results showed that Dwell was effective in improving N efficiency, as determined by yield and N content of plants, with all three soils and with both fertilizers. Higher yields were obtained when AS was used in all soils. Higher response to Dwell, but lower yields, were observed in urea treatments as compared to AS treatments. Dwell resulted in yield increase of 139 and 60% with urea in Vinton and Laveen soils, respectively, while with AS, the increases were 74 and 11%. The lowest response to Dwell occurred with the Anthony soil. Generally, higher yield was obtained when AS was thoroughly mixed and when urea was banded. Yield and N content in plants grown in these soils were in the following order: Laveen > Anthony > Vinton. Dwell was effective with both N rates, particularly with the high N rate of urea. Incubation studies showed that Dwell was effective in retarding nitrification for about 10 days. Accordingly, Dwell seems to have had no residual effect on soil nitrogen.

Knowledge of the relationship between rate of nitrification and nitrous oxide (N2O) emission, and between cumulative nitrification and N2O emission is important for developing N2O emission mitigation strategies. Gross nitrification and N2O from nitrification were determined using 15N labelling of inorganic N. N-Serve was added to delay nitrification and results showed an increase in rate of N2O emission with that of apparent nitrification in absence of N-Serve, but there was no relation in its presence. Same amount of cumulative N2O was emitted for same amount of nitrogen (N) apparently nitrified, regardless of N-Serve addition. There was no relation between N2O emission attributed to nitrification and gross nitrification with and without N-Serve. Again, same amount of cumulative N2O was emitted for same amount of gross nitrified N, regardless of N-Serve addition. These results imply that the amount of N nitrified dictates eventual cumulative N2O emitted, regardless of rate of nitrification. / February 2017

Nitrification is central to the global cycling of nitrogen and is important for primary production and other processes in marine systems, where nitrogen is generally the limiting nutrient. The majority of studies on nitrification, however, have focused on terrestrial systems, where this process can cause significant losses of N-based fertilisers from agricultural land. Comparatively little is known of nitrification in marine systems and the organisms involved. In addition, technical difficulties in isolation of pure cultures of ammonia-oxidising bacteria have severely restricted studies of species diversity and community structure of these organisms. In this study, molecular technology, based on the 16S ribosomal RNA (rRNA) molecule was applied to the characterisation of marine communities of ammonia-oxidising bacteria. PCR primers were designed for specific amplification of the rRNA gene (rDNA) from ammonia-oxidisers belonging to the beta-subdivision of the Proteobacteria. These primers were used to characterise both enrichment cultures and communities within polluted and unpolluted sediment samples. These studies indicated considerable diversity of beta-subgroup ammonia-oxidisers within marine systems which has not previously been detected. It was also shown that enrichment and isolation techniques select for strains belonging to the genus Nitrosomonas while the majority of sequences obtained by direct analysis of rDNA amplified from total genomic extracts belonged to the genus Nitrosospira. In addition, novel isolation methods were developed which considerably reduced the level of heterotrophic contamination and greatly facilitated isolation of pure cultures. In situ probing, using fluorescently labelled rRNA oligonucleotide probes, indicated that CCD microscopy is less sensitive than UV microscopy alone due to quenching of the signal.

Thesis (Master of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Wastewater treatment methods have always gravitated towards the use of biological methods for the treatment of domestic grey water. This has been proven to offer a series of advantages such as the reduction of pollution attributed to the use of synthetic chemicals; therefore, this decreases the requirement of further costly post primary treatment methods. Although such biological methods have been used for decades, their efficiency and sustainability has always been challenged by inhibitory toxicants which renders the systems redundant when these toxins are prevalent in high concentrations, culminating in the deactivation of biomass which facilitates the treatment. In most instances, this biomass is anaerobic sludge. Hence, the proposal to utilize biofilms which are ubiquitous and selfsustaining in nature. The use of engineered biofilms in wastewater treatment and their behaviour has been studied extensively, with current research studies focusing on reducing plant footprint, energy intensity and minimal usage of supplementary synthetic chemicals. An example of such processes include traditional nitrification and denitrification systems, which are currently developed as simultaneous nitrification and aerobic denitrification systems, i.e. in a single stage system, from the historical two stage systems. However, there is limited literature on biofilm robustness against a potpourri of toxicants commonly found in wastewater; particularly for total nitrogen removal systems such as simultaneous nitrification and denitrification (SND). This study was undertaken (aim) to assess the ability of biosurfactant producing biofilms in the removal of total nitrogen in the presence of toxicants, i.e. heavy metals and phenol, as biosurfactants have been proven to facilitate better mass transfer for pollutant mitigation. Unlike in conventional studies, the assessment of biosurfactant producers in total nitrogen removal was assessed in both planktonic and biofilm state. Since biofilms are known to have increased tolerance to toxic environmental conditions, they were developed thus engineered using microorganisms isolated from various sources, mainly waste material including wastewater as suggested in literature reviewed, to harness microorganisms’ possessing specified traits that can be developed when organisms are growing under strenuous environments whereby they are tolerant to toxic compounds. The assessment of these engineered biofilms involved the development from individual microorganisms to form biofilms in 1L batch reactors where the isolated microorganisms were grown in basal media containing immobilisation surfaces. The assessment of the total nitrogen efficiency was conducted using Erlenmeyer flasks (500mL) in a shaker incubator, with the biofilm TN removal efficiency being assessed in batch systems to ascertain simultaneous nitrification and denitrification rates even in the presence of heavy metals (Cu2+, Zn2+) and C6H5OH. Ambient temperature and dissolved oxygen conditions were kept constant throughout the duration of biofilm development with microorganisms (initially n = 20) being isolated for the initiation of biosurfactant studies which included screening. Results indicated that the engineered biofilms, constituted by biosurfactant producing organisms (n = 9), were consisiting of bacteria (97.19%), Protozoa (2.81%) and Archaea (0.1%) as identified using metagenomics methods. Some of the biosurfactant produced had the following functional group characteristics as determined by FTIR: -CH3-CH2, deformed NH, -CH3 amide bond, C-O, C=O, O-C-O of carboxylic acids, and C-O-C of polysaccharides. Other selected microorganisms (n = 5) tolerated maximum concentrations of the selected toxicants (Cu2+, Zn2+ and C6H5OH) of 2400 mg/L, 1800 mg/L and 850 mg/L, respectively. Enzyme analysis of the total nitrogen removal experiments indicated a higher nitrogen removal rate to be the Alcanigene sp. at 180 mg/L/h.

Efluentes com grandes concentrações de fenol e nitrogênio amoniacal apresentam grande potencial poluidor ao meio ambiente. Exemplo deste tipo de despejo é o da unidade de coqueria da indústria siderúrgica que apresenta em termos quantitativos os fenóis como os principais constituintes orgânicos. Constituintes inorgânicos também estão presentes neste tipo de despejo e são principalmente cianeto, tiocianato, sulfato e nitrogênio amoniacal, sendo que a concentração deste último pode atingir níveis de centenas de miligramas por litro. No Estado de São Paulo, para atendimento às legislações em vigor - Decreto Estadual 8468/76 e Resolução CONAMA 357/05 - em termos de nitrogênio amoniacal e fenóis, as fontes poluidoras devem atender aos limites de emissão de 20 mg N/L para nitrogênio amoniacal total e 0,5 mg C6H5OH/L para índice de fenóis, e além disso, devem atender a classificação do corpo d\'água. Após resultados bem sucedidos de pesquisas realizadas anteriormente no Departamento de Engenharia Hidráulica e Sanitária da EPUSP objetivando remoção de compostos fenólicos e nitrogênio amoniacal, presentes em uma água residuária sintética de coqueria, através de nitrificação/desnitrificação do afluente em sistemas de lodo ativado, idealizou-se a presente pesquisa objetivando promover a nitrificação/desnitrificação de um despejo similar contendo altas concentrações de fenol (1000 mg/L) e nitrogênio amoniacal (500 a 1000 mgN/L) em sistema piloto de lodo ativado de lodo único, com duas concepções distintas: parcial (\"P\") a nitrito (ou \"nitritação/desnitritação\") em um primeiro caso, somente com fonte interna de carbono para a desnitrificação, e total (\"T\") num segundo caso, com fontes interna e externa de carbono. Além disso, também foi objetivo comparar os resultados obtidos do sistema piloto \"P\" com um sistema em bateladas seqüenciais operado paralelamente para remoção de nitrogênio via nitrito. A pesquisa permitiu concluir que o sistema contínuo, em virtude de seu regime operacional, não foi eficiente para remoção de nitrogênio via nitrito por não favorecer a manutenção de amônia livre na fase aeróbia em concentrações inibitórias aos microrganismos oxidadores de nitrito, já que segundo os resultados satisfatórios do sistema em bateladas seqüenciais, a existência de amônia livre e o pH se mostraram os principais parâmetros que regem o acúmulo de nitrito no reator. Apesar disso, o sistema apresentou resultados satisfatórios quanto à desnitrificação com fenol como fonte de carbono sendo que, durante toda a operação do sistema, o efluente final apresentou concentrações de fenol desprezíveis. Resumindo, o sistema contínuo só se mostrou adequado para remoção de nitrogênio via nitrato, ao passo que o sistema em batelada favoreceu a remoção via nitrito. Quanto ao sistema \"T\", que visava nitrificação/desnitrificação completa com fontes interna e externa de carbono, os resultados permitiram concluir que, apesar do etanol ser utilizado com eficiência pelas bactérias heterotróficas para promover a desnitrificação, seu uso como fonte externa de carbono não foi adequado em sistema de lodo único. Isto porque os microrganismos deixaram de utilizar o fenol na desnitrificação passando a utilizar somente o etanol, provocando acúmulo de fenol e desequilíbrio do sistema. Sendo assim, concluiu-se que o uso de etanol como fonte externa de carbono para a desnitrificação só seria recomendável em reator anóxico em separado, ou seja, em sistema de dois lodos e não de lodo único, como o da presente pesquisa. / Wastewaters containing high phenol and ammonium concentrations present a great pollutant potential to the environment. An example of this kind of effluent is the discharge of coke-plants, which presents, quantitatively, the phenols as the main organic compound. Inorganic compounds are also present in these wastewaters and are mainly cyanide, thiocyanate, sulphate and ammonium, the last one being able to achieve hundreds of milligrammes per litre. In Sao Paulo State, there are two legislations to be accomplished, State Decree 8468/76 and CONAMA Resolution 357/05 that stand that the polluters must accomplish the discharge limits of 20 mg N/L to total ammonium nitrogen and 0,5 mg C6H5OH/L to phenols, as well as accomplish the waterbody classification. The present research was planned after well succeded results of former researches in EPUSP\'s Hydraulic and Sanitation Engineering Department aiming to remove phenolic compounds and ammonium from a synthetic coke-plant wastewater, by nitrification/denitrification of the influent in activated sludge plants. The main purpose of this work was to remove nitrogen of a similar wastewater containing high phenol (1000 mg/L) and ammonium (500 a 1000 mgN/L) concentrations in two activated sludge pilot plants (single sludge): a partial one (\"P\") to remove nitrite (or \"nitritation/denitritation\") in the first case, only with internal carbon source for the denitrification, and a total one (\"T\") in a second case, with internal and external carbon sources. It was also aim of this work to compare the results obtained by the Partial (\"P\") pilot system with a parallel batch sequence reactor operated to remove nitrogen via nitrite. The research concluded that the continuos system, due to its operational characteristic, was not efficient to remove nitrogen via nitrite that does not favor the maintenance of free ammonia in the oxic phase in inhibitory concentrations to the nitrite oxidizers, as according to the wellsucceeded batch system, the existance of free ammonia and the pH have been the main parameters to raise the nitrite accumulation in the reactor. Nevertheless, the system presented satisfactory results to the denitrification with phenol as carbon source and, during the whole experimental work, the final effluent just showed despicable phenol concentrations. Summing up, continuos system was just adequate to remove nitrogen via nitrate, while the batch system favored its removal via nitrite. Due to the Total (\"T\") system which aims to complete nitrification/denitrification with internal and external carbon sources, the results showed that, despite ethanol having been successfully used by the heterotrophic bacteria to denitrification, its use as external carbon source was not adequate in single sludge system, because the microorganisms do not use phenol in denitrification, just using ethanol, causing phenol accumulation and unbalance of the system. Therefore, it was concluded that the use of ethanol as external carbon source to denitrification would be recommended only in anoxic separated reactor, i.e., in a double sludge system and not in a single sludge one, as this research.