Ce travail réalise une synthèse des connaissances du programme de recherche PIREN Seine concernant la problématique de l'azote sur le bassin du Grand Morin (1200 km2). A cet effet, la plate-forme de modélisation CAWAQS (CAtchment WAter Quality Simulator) a été construite. CAWAQS simule la qualité de l'eau des différents compartiments d'un bassin versant: sols, aquifères, cours d'eau. Il a été construit à partir des modèles STICS, NEWSAM et ProSe. L'utilisation conjointe de techniques géostatistiques (logiciel Isatis®) et de CAWAQS a permis de dresser un état de la contamination en nitrate des aquifères sur la période 1977-1996. La confrontation des concentrations de nitrate simulées en rivière avec des observations à haute fréquence sur la période 1991-1996 permet l'estimation des capacités dénitrifiantes. On estime sur la période 1991-1996, que les nitrates infiltrés à la base de la zone racinaire (4 000 tN.an-1) sont exportés vers l'aval par le réseau hydrographique (41 %), stockés dans les aquifères (39%) et éliminés par dénitrification (20 %) dans les zones humides, les nappes alluviales, la zone hyporhéique, et par les processus benthiques en rivière. Des campagnes de terrain et des simulations numériques ont aussi été réalisées à l'aval du Grand Morin afin de prendre en compte les processus dominants dans la transformation des polluants pour les cours d'eau à faible profondeur. Les résultats obtenus suggèrent l'existence d'une forte élimination de l'azote ammoniacal par nitrification-dénitrification dans les compartiments benthiques. Pour l'ensemble du Grand Morin, la quantité d'azote éliminée par ce processus a été estimée à 140 tN.an-1.

L'objectif de ce travail était de caractériser les émissions de N2O par dénitrification et nitrification dans un sol de limon à l'échelle de la motte. La dénitrification a été étudiée par des mesures faites sur des boues de sol et une simulation des populations microbiennes réductrices de NO3- et N2O. La capacité réductrice de N2O est inductible, de sorte que la production nette de N2O diminue fortement au cours du temps. La respiration et l'émission de N2O par dénitrification par des mottes de sol étaient fortement stimulées par une pré-incubation anaérobie. Ces résultats peuvent être expliqués à l'aide d'un modèle simplifié prenant en compte la structure des mottes. La vitesse de nitrification et sa contribution à la production de N2O ont été caractérisées sur des agrégats de sol en fonction de la pression partielle en O2, en utilisant le traçage 15N. Une réduction de la pression O2 diminue la vitesse de nitrification et augmente fortement la production de N2O par nitrification

L'objectif principal de ce travail a porté sur l'optimisation du dimensionnement et de la gestion des installations de traitement des eaux usées par boues activées fonctionnant à basse température (10-12°C) et soumise à une contrainte de traitement poussé sur l'azote. Dans ce but, nous avons étudié la faisabilité de la nitrification à des charges massiques stabilisées supérieures à 0.1 kgDBO5.(kgMVS.j)-1. Le fonctionnement d'une installation pilote alimentée par un effluent synthétique, puis par un effluent urbain réel, a été étudié pour différentes charges massiques stabilisées à une température de 11 +/- 1°C. Les résultats très détaillés acquis en continu sur deux périodes de six mois ont suggéré le rôle et la hiérarchie des facteurs influençant la capacité de nitrification. Ils ont permis d'adapter l'outil de simulation dynamique Activated Sludge Model n°1 (ASM1). La double démarche expérimentale et numérique mise en oeuvre a permis de dégager les rôles des conditions de fonctionnement (âge des boues, charge volumique en azote, durée de présence d'oxygène) et des propriétés de l'effluent à traiter (ratios caractéristiques, part des différentes fractions) sur les performances de traitement de l'azote et sur la composition de la boue. Les valeurs limites des paramètres de gestion (charge massique maximale, durée d'aération journalière), permettant à différentes configurations d'installation (bassin unique, réacteur avec zone d'anoxie en tête) de respecter une concentration en azote total inférieure à 10 mgN/L dans le rejet, ont pu être dégagées. Des stratégies de gestion dans le cas de variations de flux importantes en entrée d'installation (cas des zones touristiques de montagnes) ont pu être évaluées (fonctionnement à charge massique variable par adaptation de la durée d'aération, ou bien utilisation d'un bassin d'aération à volume variable).

La prise de conscience de l'importance de la gestion des déchets et de la préservation des ressources a conduit de plus en plus d'acteurs du monde agricole à s'intéresser au compostage comme mode de traitement d'effluents d'élevage ou de boues de stations d'épuration. Les filières d'utilisation de ces substrats, épandage, compostage ou autre, doivent donc être comparées. On sait en effet que l'agriculture est responsable d'une part importante des émissions d'ammoniac (NH3) et de protoxyde d'azote (N2O) et ainsi, indirectement ou directement, de l'augmentation de la concentration de N2O dans l'atmosphère. Trois séries d'expérimentations ont été menées respectivement sur des mélanges d'écorces de peuplier et de fientes de poules pondeuses, sur du fumier de poulet sans ou avec additifs et sur un mélange de paille et de boues de station d'épuration rurale, en parallèle avec un nouveau mélange d'écorces et de fientes. La première a permis de mesurer les émissions de gaz azotés d'un andain (25 m3) en cours de compostage. Elles étaient de 50% environ de l'azote initial sous forme N- NH3 et de moins de 1% sous forme N-N2O. La production de N2O était associée à des activités de nitrification/dénitrification et avait principalement lieu en phase de maturation. La seconde a révélé, pour du compostage de courte durée (1,5 mois) et en taille expérimentale (2 m3), les conditions dans lesquelles les émissions de NH3 et de N2O étaient les plus fortes ou les plus faibles et lesquelles favorisaient l'activité de dénitrification des micro-organismes. L'augmentation du rapport carbone/azote et l'addition de composés ou microorganismes adéquats font passer les pertes en NNH3 de 55 à 7% de l'azote initial. Leur effet sur la libération de N2O (0,2-0,9%) dépend de nombreux facteurs. La troisième a montré que les andains de compostage (10 et 25 m3) devaient être considérés comme des écosystèmes à part entière composés de différents compartiments (grossièrement entrée, fond, sortie et croûte) où les populations microbiennes s'organisent différemment, l'entrée étant productrice de N2O, le fond et la sortie, producteurs, en équilibre ou consommateurs suivant les moments. La taille de l'andain en expérimentation est donc une donnée capitale pour considérer les résultats comme représentatifs de la réalité.

<p>Two ecological models of nitrogen processes in treatment wetlands have been evaluated and compared. These models have been implemented, simulated, and visualized in the Modelica language. The differences and similarities between the Modelica modeling environment used in this thesis and other environments or tools for ecological modeling have been evaluated. The modeling tools evaluated are PowerSim, Simile, Stella, the MathModelica Model Editor, and WEST. </p><p>The evaluation and the analysis have been performed using McCall’s factors for software quality (McCall et al, 1977), a correlation analysis and the Constant Comparative Method (Glaser&Strauss, 1999). The results show that the modeling tools and the models can both be separated into two categories: Simple Components and Complex Components for the modeling tools, and Simple Models and Complex Models for the models. The major difference between the Simple Components and the Complex Components is the higher possibility of the Complex Components to create and reuse separate components and the higher complexity in these components. The similarities between the categories are that they are consistent, easy to overview and use, if no new components are to be created. The major difference between the Simple Models and the Complex models lies in the number of functions and in the possibility of reuse and expansion. The similarities between all the models are that they are all consequent, logical, valid, specialized, and easy to use if the user has programming skill. </p><p>To conclude thisthesis, the nitrogen decrease in a constructed treatment wetland can well be simulated using the Nitrification/Denitrification model expressed in Modelica and the MathModelica Model Editor. However, some changes to the Model Editor are recommended to make the creation of the model easier. The most important of these changes are the addition of a tutorial, the ddition of useful error handling and messages, and the removal of unnecessary Visio features.</p>

This research investigates the physiological and transcriptional responses of Nitrosomonas europaea when exposed to chlorinated aromatic compounds and heavy metals under varying environmental conditions. It was found that transcriptional responses of identified sentinel genes correlate well with nitrification inhibition. Sorption of metals to biomass was also investigated and found not to correlate well with N. europaea inhibition. Whole genome microarray experiments were performed to define the transcriptional response of N. europaea when exposed to chlorobenzene. 13 out of 2460 N. europaea genes were significantly up-regulated after a 1-hour exposure to 4 μM chlorobenzene. HPLC analysis revealed that chlorobenzene was being oxidized primarily into 4-chlorophenol, and further physiological studies revealed that the presence of 4-chlorophenol could account for the inhibitory responses observed. RT-qPCR analysis of several differentially regulated genes verified that similar transcriptional responses were occurring for both chlorobenzene and 4-chlorophenol. 50% inhibitory concentrations of chlorobenzene and 4-chlorophenol resulted in moderate up-regulation of studied genes, however, increasing the concentration of 4-chlorophenol to achieve nitrification inhibition of 93% or more dramatically increased the fold regulation of several of the identified up- and down- regulated genes of interest. Increasing the 4-chlorophenol exposure time to 3 hours at the higher inhibition levels led to a general decrease in amplitude of transcriptional response for all genes tested. Cultures of N. europaea were exposed to various amounts of cadmium in aqueous solution containing EDTA, a strong metal-chelating organic, to control free ionic cadmium²⁺ (Cd²⁺) concentrations. Inhibition of ammonia oxidation as well as transcriptional up-regulation of merA, an identified sentinel gene for exposure to cadmium was found to correlate well with the concentration of Cd²⁺. The concentration of Cd²⁺ required to significantly affect N. europaea cells was found to be in the nanomolar range, which is several orders of magnitude lower than values reported in the literature for cadmium inhibition to mixed-culture activated sludge systems. The sorption of cadmium to the cells was found to be proportional to both the concentration of total cadmium and the concentration of Cd²⁺. At the concentration of metals required to cause approximately 50% nitrification inhibition, specific oxygen uptake results indicate the inhibition is specific to AMO with HAO and downstream energy-generation processes intact. To investigate more closely the inhibitory interactions between heavy metals and AMO, N. europaea inhibition by cadmium, zinc and silver was studied under substrate-limiting conditions. Unlike incubation in oxic environments, 1 hour incubations of N. europaea with cadmium and silver under anoxic conditions did not cause inhibition of nitrification activity after re-suspension in oxic media. In contrast, zinc, which is normally considered an analogue of cadmium in terms of toxic effect and transport mechanisms, was non-inhibitory to N. europaea when exposed in media lacking ammonia. Transcriptional response of merA closely followed the inhibition patterns, with samples which were inhibited after the removal of the metal having significant up-regulation of the gene, and those samples which were uninhibited showing no significant change in merA transcript levels compared to controls. Although sorbed metal concentrations were not found to be predictive of either extent of inhibition or transcriptional response, significantly more cadmium, zinc and silver were sorbed to biomass when incubated in aerobic media compared to anoxic media. Sorption in oxic media was found to be independent of AMO activity and similar results were obtained using Deinococcus radiodurans, a non-nitrifying gram-positive extremophile. The results indicate that greater heavy metal sorption to biomass in oxic environments may be due to general membrane chemistry effects. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from March 31, 2011 - March 31, 2012

In this thesis, the application of molecular microbiology methods to understand wastewater treatment bio-reactions is described. Two different wastewater treatment systems were chosen for the experimental work. Firstly; the activated sludge processes at two different facilities in Sweden (Västerås and Eskilstuna) were investigated and compared in a context where low temperatures can affect the efficiency of the nitrogen removal performance in terms of nitrification. Initially, fluorescence in situ hybridization (FISH) was utilised in order to quantify some of the species involved in ammonia and nitrite oxidation at Västerås, providing information on how the different communities react to decreasing temperatures. Then, the polymerase chain reaction (PCR), cloning-sequencing method was employed in order to study the composition of the ammonia oxidizing bacteria (AOB) community at the same two wastewater treatment plants (WWTPs). Secondly; the potential use of constructed wetlands for the treatment of winery wastewater was studied. High ethanol concentration artificial wastewater with and without inorganic nutrients (nitrates and phosphates) was fed in a set of pilot-scale constructed wetlands. Pollutant removal performance and enzyme activity tests were carried out. Additionally, the bacterial community structure was investigated by means of denaturing gradient gel electrophoresis (DGGE). In the first set of studies it was shown that the AOB population which plays a major role in nitrifying reactors presented a seasonal shift and a higher diversity at Västerås during winter time, while the nitrification performance maintained stable levels and the ammonia removal efficiency increased. Thus, the higher ammonia removal efficiency at Västerås could be related to the diversity of the AOB population composition. Lastly, when constructed wetlands were in focus, the differential effects of ethanol and nutrients over the chemical oxygen demand (COD) removal performance were proven. In fact, the addition of nutrients on one of the experimental wetlands increased the COD (ethanol) removal and supported the maintenance of a bacterial population similar to the control wetland (no ethanol added). In conclusion, both studies proved a strong relationship between process performance (pollution removal) and the dynamics of the bacterial communities involved.

Decreasing the nitrogen transport from land to surrounding seas is a major task throughout the world to limit eutrophication of the coastal areas. Several approaches are currently used, including the establishment of wetlands, to decrease the transport of nitrogen. Wetlands represent ecosystems where the nitrogen removal from water can be efficient given that they are appropriately designed. The aim of this thesis was to investigate and quantify the effect of critical factors that regulate the nitrogen removal in wetlands, and to develop better guidelines for wetland design. Studies were performed at different scales, from microcosms to full scale wetlands, and methods included modelling, mass balance calculations and process studies. A first order rate model was used to simulate the nitrogen transformations in two large wetlands treating wastewater containing both ammonium and nitrate nitrogen. It was found that the dynamics of the main itrogen transformation processes could not be satisfactorily described using this approach. Large wetlands containing vegetation are complex ecosystems, and the process rates vary in both time and space. The great diversity of microenvironments favours different nitrogen processes, and large differences in potential nitrification and denitrification rates were found between different surface structures within a wetland. The results from microcosms measurements showed that the highest potential for nitrification was on surfaces in the water column, while the denitrification capacity was highest in the sediment. For the sediment denitrification capacity, the plant community composition was shown to be of major importance primarily by supplying litter serving as a carbon and energy source, and/or attachment surfaces, for denitrifying bacteria. Denitrification rates may be affected more than three fold by different types of litter and detritus in the sediments. Intact sediment cores from stands of the emergent plants Glyceria maxima and Typha latifolia had higher denitrification potential than sediment cores from stands of the submersed plant Potamogeton pectinatus. However, the quality of the organic material for the denitrifying bacteria was highest in G. maxima and P. pectinatus stands. All sediment cores from the wetland were limited by carbon, and the lower denitrification capacity of the submersed plant, P. pectinatus, was likely due to lower amounts of organic matter. However, in another wetland, intact cores from stands of the submersed plant Elodea canadensis had a higher denitrification capacity than the cores from stands of T. latifolia and Phragmites australis. This was possibly due to a larger biomass, and better quality, of the organic matter from that submersed specie, or to epiphytic biofilms on the living plants. Those microcosms studies showed that both the quality of the organic matter as a substrate for the microbial communities, and the amount of organic material produced were important for the denitrification capacity. In pilot scale wetlands, the composition of the plant community was also a more important factor for high nitrate removal than the differences in hydraulic loads (equivalent of 1 or 3 d retention time), despite the cold climate. The greatest removal was found in wetlands with emergent vegetation dominated by P. australis and G. maxima, rather than in wetlands with submersed vegetation. In brief, the results presented in this thesis emphasize the importance of dense emergent vegetation for high annual nitrate removal in treatment wetlands.

The effectiveness of partial ozonation of return activated sludge for enhancing denitrification and waste sludge minimization were examined. A pair of nitrifying sequencing batch reactors was operated in either aerobic or alternating anoxic/aerobic conditions, with one control and one ozonated reactor in each set. The amount of solids decreased with the ozone dose. Biomass in the anoxic/aerobic reactor was easier to destroy than in the aerobic one, generating approximately twice as much soluble chemical oxygen demand (COD) by cell lysis. Increased COD favoured production of extracellular polymers in ozonated reactors, enhancing flocculation and improving settling. Floc stability was also strengthened in prolonged operation in alternating treatment, resulting in declined solids destruction. Dewaterability was better in alternating reactors than in aerobic ones indicating that incorporation of an anoxic zone for biological nutrient removal leads to improvement in sludge dewatering. The negative impact of ozonation on dewaterability was minimal in terms of the long-term operation. Ozone successively destroyed indicator estrogenic compounds, contributing to total estrogen removal from wastewater. Denitrification rate improved up to 60% due to additional carbon released by ozonation. Nitrification rates deteriorated much more in the aerobic than in the alternating reactor, possibly as a result of competition created by growth of heterotrophs receiving the additional COD. Overall, ozonation provided the expected benefits and had less negative impacts on processes in the alternating treatment, although after prolonged operation, benefits could become less significant. The alternating anoxic/aerobic reactor achieved twice the nitrification rates of its aerobic counterpart. Higher removal rates of estrogens were associated with higher nitrification rates, supporting the contention that the nitrifying biomass was responsible for their removal. The alternating treatment offered the better estrogen biodegradation. Microbial populations in both reactors were examined with fluorescent in situ hybridization. Dominance of rapid nitrifiers like Nitrosomonas and Nitrobacter (79.5%) in the alternating reactor, compared to a dominance of slower nitrifiers like Nitrosospira and Nitrospira (78.2%) in the aerobic reactor were found. The findings are important to design engineers, as reactors are typically designed based on nitrifiers’ growth rate determined in strictly aerobic conditions. / October 2008

The biogenic reduction of Mn(IV) oxides is one of the most favorable anaerobic electron transfer processes in aquatic systems and likely plays an important role in the redox cycle of both carbon and nitrogen in anaerobic environments; yet, the different pathways involved in the microbial transformation of Mn(IV) oxides remain unclear. The coupling between the reduction of Mn(IV) to Mn(II) and the oxidation of organic carbon to CO₂ is largely catalyzed by microorganisms in various environments such as redox stratified water columns and sediments. The recent discovery that soluble Mn(III) exists in natural systems and is formed during biological oxidation of Mn(II) implies the possibility that Mn(III) is formed as an intermediate during the microbial reduction of Mn(IV). In this dissertation, mutagenesis studies and kinetic analysis were combined to study the mechanism of microbial reduction of Mn(IV) by Shewanella oneidensis MR-1, one of the most studied metal-respiring prokaryotes. We show for the first time that the microbial reduction of Mn(IV) proceeds step-wise via two successive one-electron transfer reactions with soluble Mn(III) as intermediate produced in solution. The point mutant strain Mn3, generated via random chemical mutagenesis, presents a unique phenotype that reduces solid Mn(IV) to Mn(III) but not to Mn(II), suggesting that these two reduction steps proceed via different electron transport pathways. Mutagenesis studies on various in-frame deletion mutant strains demonstrate that the reduction of both solid Mn(IV) and soluble Mn(III) occurs at the outer membrane of the cell and Mn(IV) respiration involves only one of the two potential terminal reductases (c-type cytochrome MtrC and OmcA) involved in Fe(III) respiration. Interestingly, only the second electron transfer step is coupled to the respiration of organic carbon, which opposes the long-standing paradigm that microbial reduction of Mn(IV) proceeds via the single transfer of two electrons coupled to the mineralization of carbon substrates. The coupling between anaerobic nitrification and Mn reduction has been demonstrated to be thermodynamically favorable. However, the existence of this process in natural system is still in debate. In this dissertation, characterization of coastal marine sediments was combined with laboratory incubations of the same sediments to investigate the effect of Mn oxides on the redox cycle of nitrogen. Our slurry incubations demonstrate that anaerobic nitrification is catalyzed by Mn oxides. In addition, mass balance calculations on NH₄⁺ link the consumption of NH₄⁺ to anaerobic ammonium oxidation in the presence of Mn oxides and confirm the occurrence of Mn(IV)-catalyzed anaerobic nitrification. The activity of anaerobic nitrification is greatly affected by the initial ratio of Mn(IV) to NH₄⁺, the reactivity of Mn oxides, and the reducing potential of the system. Overall, Mn(IV)-catalyzed anaerobic nitrification may be an important source of nitrite/nitrate in anaerobic marine sediments and provide an alternative pathway for subsequent nitrogen losses in the marine nitrogen cycle.