La respiration anaérobie peut être un trait essentiel dans le mode de vie, la colonisation de l'environnement et la survie. Jusqu'à présent, la seule respiration anaérobie confirmée chez Agrobacterium spp. est la dénitrification. De façon intéressante, cette voie est inégalement répandue chez les agrobactéries. Ces observations m'ont amené à mon hypothèse, à savoir la respiration anaérobie et notamment la dénitrification pourraient expliquer la coexistence d'agrobactéries et leur distribution dans des niches spécifiques de la rhizosphère. Ma thèse visait à explorer les stratégies de respiration anaérobie d’Agrobacterium spp. et de les relier à l'adaptation de niche écologiques différentes. Les objectifs de ma thèse étaient (1) de caractériser tous les gènes impliqués dans la dénitrification chez A. fabrum C58 in vitro, (2) d'explorer les gènes de la dénitrification nécessaires à la colonisation des racines du maïs et (3) de découvrir de nouvelles respirations anaérobies pendant la colonisation racinaire du maïs (Figure 16). Réaliser des mutants et les tester dans des conditions particulières est le moyen classique de déterminer l'implication d'un gène dans une voie spécifique. Cependant, cette méthode implique une vision à priori et des connaissances solides sur les gènes cibles et ne peut pas être appliquée à toutes les situations. Nous avons alors dû développer une méthode plus adaptée pour identifier les gènes essentiels impliqués dans la croissance dans des conditions anaérobies spécifiques. - Gènes de dénitrification chez A. fabrum C58 in vitro. Pour compléter la voie de dénitrification chez A. fabrum C58 et identifier tous les gènes et régulateurs impliqués dans la dénitrification, nous avons adopté deux stratégies : Premièrement, une vision à priori pour (1) identifier la nitrate réductase impliquée dans la première étape de la dénitrification et (2) valider le rôle d'un ARN non codant dans le contrôle de la dénitrification. Pour ce faire, nous avons construit un mutant napA de A. fabrum C58 et un mutant de l'ARN non codant NopR et nous avons évalué leur croissance et leur capacité à produire du N2O dans des conditions anoxiques. Deuxièmement, pour identifier tous les gènes impliqués dans la dénitrification, nous avons construit une banque de transposons mutants de C58 et testé sa croissance dans des conditions de dénitrification in vitro en présence de nitrate ou de nitrite. - Rôle des gènes de la denitrification de A. fabrum C58 dans la colonisation racinaire du maïs. Il est bien connu que le séquençage de transposons (Tn-Seq) est une méthode très puissante pour déterminer les gènes nécessaires à la croissance bactérienne en présence de leur hôte. Pour déterminer les gènes de dénitrification impliqués dans la colonisation des racines en anoxie, nous avons utilisé la banque construite chez C58 et l’avons inoculée sur les plants de maï cultivées sur un sol fertile et cultivées dans des conditions inondées mimant des conditions anaérobies. Le séquençage des cellules d ‘A. fabrum C58 récupérées mettra en évidence les gènes impliqués dans la colonisation anaérobie de cette niche spécifique. - Découverte de nouvelles voies de respiration anaérobie chez A. fabrum C58. Pour découvrir de nouvelles voies de respiration anaérobie, nous avons mis en place des tests de croissance de C58 dans des conditions anoxiques en présence de sources de C et de N en tant qu'accepteurs terminaux d'électrons. De façon interéssante, en cultivant des souches WT et mutée dans le gène napA au contact de la racine de maïs dans des conditions anoxiques (chapitre 1), nous avons montré une croissance des deux souches. Ce résultats suggère que les exsudats de racine servent d'accepteurs d'électrons terminaux pour la croissance anaérobie de C58. Pour déterminer quels composés exsudés du maïs peuvent servir de TEA, les principaux métabolites ont été identifiés par HPLC et certains ont été testés en tant que TEA dans des conditions anoxiques / Anaerobic respiration may be an essential trait in lifestyle, environment colonization and survival. Until now, the only confirmed anaerobic respiration in Agrobacterium spp. is denitrification. Interestingly, this pathway is unequally widespread among Agrobacteria. These observations led me to my hypothesis which is anaerobic respiration and notably denitrification could explain the coexistence of Agrobacteria and their distribution in specific niches in the rhizosphere. My thesis was undertaken to explore the anaerobic respiration strategies of Agrobacterium spp. and to relate them to niche adaptation. The objectives of my thesis were to (1) characterize all the genes involved in denitrification in A. fabrum C58 in vitro, (2) explore the genes of denitrification that are needed during maize root colonization and (3) discover new anaerobic respirations that occur during maize root colonization (Figure 16). Mutational analysis is the classic way to determine the involvement of a gene in specific pathway. However, this method implies an a priori view and solid knowledge on target genes and cannot be applied for every situation. We have to develop a more adapted method to identify essential genes involved in growth in specific anaerobic conditions. - Denitrification genes in A. fabrum C58 in vitro. To complete denitrification pathway in A. fabrum C58 and identify all the genes and regulators involved in the denitrification function, we adopted two strategies: Firstly, an a priori view to (1) identify the nitrate reductase involved in the first step of denitrification and (2) validate the role of a non-coding RNA in denitrification control. To do so, we constructed a mutant of napA of A. fabrum C58 and a mutant of the non-coding RNA NopR and we evaluated their growth and capacity to produce N2O under anoxic conditions. Secondly, to identify all the genes involved in denitrification, we constructed a mutant transposon library of C58 and tested its growth under denitrification conditions in vitro in the presence of either nitrate or nitrite. - Role of A. fabrum C58 denitrifying genes in the root colonization of maize. It is well known that Transposon-sequencing (Tn-Seq) is a very powerful method to determine genes required for bacterial growth in the presence of their host. To determine denitrifying genes involved in root colonization under anaerobic conditions, we used the library constructed in C58 and performed in planta assays. The mutant library was inoculated on maize plants grown on fertile-ground and cultured under flooded conditions miming anaerobic conditions. Sequencing the recovered A. fabrum C58 cells will evidence the genes involved in this anaerobically specific niche colonization. - Discovery of new anaerobic respiration pathways in A. fabrum C58. To discover new anaerobic respiration pathways, we set-up growth assays of C58 under anoxic conditions in the presence C and N sources as terminal electrons acceptors. Interestingly, by culturing WT and NapA-deficient strains in contact with maize root under anoxic conditions (Chapter 1), we showed growth of both strains, suggesting that root exudates serve as terminal electrons acceptors for anaerobic growth of C58. To determine which maize exuded compounds can serve as TEAs, primary metabolites were identified by HPLC and some were tested as TEAs under the set-up conditions

Thesis (Ph. D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), February 2002. / Vita. / Includes bibliographical references (leaves 153-161). / I used direct measurements of nitrogen gas (N₂ fluxes and a ¹⁵N stable isotope tracer to determine the contribution of denitrification to salt marsh sediment N cycling. Denitrification in salt marsh tidal creekbottoms is a major sink for groundwater nitrate of terrestrial origin. I studied creekbottom denitrification by direct measurements of N₂ fluxes in closed chambers against a low-N₂ background. I undertook experiments and simulation modeling of sediment N₂ fluxes in closed chambers to optimize the key experimental parameters of this approach. Denitrification in these sediments was driven by the degradation of labile organic matter pools which are depleted during long incubations. Sediment thickness was the most important parameter controlling the required incubation time. Errors of up to 13% with gas headspaces and 80% with water headspaces resulted from headspace N2 accumulation and the resulting collapse of the sediment-water diffusion gradient. These errors could be eliminated by using headspaces of sufficient thickness. Headspace flushing to reduce ammonium accumulation did not affect denitrification rates, but caused transient disturbance of N₂ flux rates. Direct measurements of 0₂, C0₂, N₂, and inorganic N fluxes from the sediments of a salt marsh tidal creek were made in order to examine the interaction of denitrification with the carbon, oxygen, and N cycles. Organic carbon concentration and lability were the primary controls on metabolic rates. C0₂/N flux ratios averaged 6.1, indicating respiration driven by algal biomass. / (cont.) Allochthonous denitrification accounted for 39% of total sediment denitrification (2.7 mol N m⁻² yr⁻¹). 46% of remineralized ammonium was denitrified, while the contribution of autochthonous denitrification to 0₂ and C0₂ fluxes was 18% and 10%, respectively. A ¹⁵N-ammonium tracer was used to study competition between plants and nitrifying bacteria for remineralized ammonium. In undisturbed sediments of Spartina alterniflora, plant uptake out-competed nitrification-denitrification, with plant uptake accounting for 66% of remineralized ammonium during the growing season. Under N fertilization (15.5 mol m⁻² yr⁻¹), both plant N uptake and denitrification increased, but denitrification dominated, accounting for 72% of the available N. When plant uptake was hydrologically suppressed, nitrification-denitrification was stimulated by the excess N, shifting the competitive balance toward denitrification. / by Michael Robert Hamersley. / Ph.D.

Plants in constructed wetlands serve as carriers for attached microbial growth. They mainly transfer oxygen and release exudates into the root zone. As a result, an area exists around the roots (rhizosphere) in which bacteria are stimulated by root growth. Our goals were to ascertain whether stimulating the microbial community only has a local effect on the rhizoplane, and to establish the importance of this stimulation for wastewater purification in the root zone. Observations were carried out in a planted and an unplanted laboratory batch reactor incubated with an artificial wastewater with a high concentration of ammonia. Samples were taken at intervals of 10 mm away from the roots. The chemical and physical conditions and enzyme activities in soil sections at various distances from the roots affecting the efficiency of microbial nitrogen removal were characterized. An influence on the nitrification and denitrification process was detected via several parameters up to a range of different root distances: microbial parameters such as the total bacterial number, respiratory activity, protein and DNA amount seem to be affected by roots up to a distance of 50 mm from the roots, whereas the oxygen concentration, DOC and redox potential are unaffected at a distance exceeding 20-30 mm. This indicates that improved nitrogen removal is also possible in the wider root surroundings. Given the average root-to-root distance of 35 mm, the root-influenced area could therefore be expanded to the whole rooted zone in a constructed wetland.

The gap between resource demand for the industrialized world and non-renewable feedstock like fossil fuels, essential agricultural fertilizer is getting increasingly severe, which has resulted in alarming-increasingly impacts on worldwide environmental problems. Meanwhile, wastewater treatment plants (WWTPs), as an essential facility for urbanization, is also confronting new rising challenges such as energy consumption and operation costs rather than only improving effluent wastewater quality. It is thereby important to develop new approaches for next-generation WWTPs with less energy consumption and costs in a sustainable way. The objective was to study the application of volatile fatty acids (VFAs) from waste as an external carbon source on denitrification by manometric tracking method in the lab-scale. Food waste and primary sludge were used for anaerobic fermentation to produce VFAs, which was then used as a sole external carbon source with various C/N ratio in denitrification batch test. The results were compared with traditional external carbon sources, acetate and methanol. It was found that the maximum denitrification rate with VFAs as an external carbon source was 15.73 ± 0.95 mg NOx-N/g VSS h, faster than those with acetate and methanol as external carbon sources. When C/N ratio ≥ 4.5 nitrate removal efficiency and the highest maximum denitrification rate were reached, the optimum C/N ratio for dosing VFAs as an external carbon source was thus determined as 4.5. In addition, denitrification capacity experiments with addition of VFAs produced from three different pH-controlled digestion reactors as an external carbon source were then conducted under an optimum C/N ratio of 4.5. As a result of the composition difference between VFAs produced from different pH environment, it was observed that, with VFAs from pH-10 digestion reactor denitrification rate was slightly higher than those with VFAs from pH-5 and none-pH-controlled digestion reactors. Furthermore, denitrification batch test using chemical tracking method was conducted to compare with manometric tracking method, and it was proven that the results obtained from manometric denitrification tests were reliable and valid. The overall results show that VFAs produced from anaerobic fermentation are an excellent external carbon source for denitrification, and it realizes the utilization of carbon resource recovery from WWTPs, which is crucial for next-generation wastewater treatment. / Klyftan mellan resursefterfrågan i den industrialiserade världen och icke-förnybar råvara som fossila bränslen, essentiell jordbruksgödsel, blir allt svårare, vilket har resulterat i oroväckande effekter på globala miljöproblem. Samtidigt står avloppsreningsverk (ARV), som är en viktig anläggning för urbanisering, inför nya ökande utmaningar som energiförbrukning och driftkostnader snarare än att förbättra kvaliteten på renat avloppsvatten. Det är därmed viktigt att utveckla nya strategier för nästa generations ARV med mindre energiförbrukning och kostnader på ett hållbart sätt. Målet var att studera tillämpningen av flyktiga fettsyror (VFA) från avfall som en extern kolkälla i denitrifikation med manometrisk metod i laboratorieskala. Matavfall och primärslam användes för anaerob rötning för att producera VFA, som sedan användes som en extern kolkälla med olika C/N-förhållanden i denitrifikationstest. Resultaten jämfördes med traditionella externa kolkällor, acetat och metanol. Det visade sig att maximal denitrifikationskapacitet med VFA som extern kolkälla var 15,73 ± 0,95 mg NOx-N/g VSS h, snabbare än de med acetat och metanol som extern kolkälla. När C/N-förhållandet ≥4,5 uppnåddes högsta nitratborttagningseffektivitet och maximala denitrifikationskapacitet, varför det optimala C/N-förhållandet för dosering av VFA som extern kolkälla bestäms som 4,5. Dessutom genomfördes experiment med denitrifikationskapacitets med tillsats av VFA producerat från tre olika pH-kontrollerade rötningsreaktorer som externa kolkällor under ett optimalt C/N-förhållande av 4,5. Som ett resultat av kompositionsskillnaden mellan VFA producerade från olika pH-miljöer, det observerades att med VFA från pH-10 kontrollerad reaktor denitrifikationskapacitet var något högre än med VFA från pH-5 och en icke-pH-kontrollerad reaktor. Vidare utfördes denitrifikationstest med användning av kemisk metod för jämförelse med manometrisk metod, och det visades att resultat erhållna från manometrisk denitrifikationstest var tillförlitliga och giltiga. De övergripande resultaten visar att VFA som produceras från anaerob rötning är en utmärkt extern kolkälla för denitrifikationsprocessen, vilket betyder att utnyttjandet av kolresursåtervinning från AVR är avgörande för nästa generations avloppsrening.

Elevated concentrations of nutrients, particularly nitrate, in groundwater and springs in Florida are a growing resource management concern. Stormwater infiltration basins, which are a common stormwater management practice in the well-drained karst terrain areas of Florida, are a potentially important source of nutrients to the groundwater system because stormwater exits the basin by only evaporation or infiltration. To better understand the biogeochemical processes integrating stormwater infiltration impacts on groundwater resources in a field-scale setting, a combination of hydrologic, soil chemistry, water chemistry, dissolved and soil gas, isotope, and microbiological data was collected from 2007 through 2010 at two stormwater infiltration basins receiving runoff from predominantly residential watersheds in north-central Florida. Substantially different biogeochemical processes affecting nitrogen fate and transport were observed beneath the two stormwater infiltration basins. Differences are related to soil textural properties that deeply link hydroclimatic conditions with soil moisture variations in a humid, subtropical climate. During 2008, shallow groundwater beneath the basin with predominantly clayey soils (median 41% silt+clay content) exhibited decreases in dissolved oxygen from 3.8 to 0.1 mg/L and decreases in nitrate-nitrogen from 2.7 mg/L to less than 0.016 mg/L, followed by manganese and iron reduction, sulfate reduction, and methanogenesis. In contrast, beneath the basin with predominantly sandy soils (median 2% silt+clay content), aerobic conditions persisted from 2007 through 2009 (dissolved oxygen of 5.0â€"7.8 mg/L), resulting in nitrate-nitrogen of 1.3â€"3.3 mg/L in shallow groundwater. Soil extractable nitrate-nitrogen was significantly lower and the copper-containing nitrite reductase gene density was significantly higher beneath the clayey basin. Differences in moisture retention capacity between fine- and coarse-textured soils resulted in median volumetric gas-phase contents of 0.04 beneath the clayey basin and 0.19 beneath the sandy basin, inhibiting surface/subsurface oxygen exchange beneath the clayey basin. Subsurface biogeochemical processes at the clayey stormwater infiltration basin were further analyzed to better understand the effects of the highly variable hydrologic conditions common in humid, subtropical climates. Cyclic variations in biogeochemical processes generally coincided with wet and dry hydroclimatic conditions. Oxidizing conditions in the subsurface persisted for about one month or less at the beginning of wet periods with dissolved oxygen and nitrate showing similar temporal patterns. Reducing conditions in the subsurface evolved during prolonged flooding of the basin. At about the same time oxygen and nitrate reduction concluded, manganese, iron, and sulfate reduction began, with the onset of methanogenesis one month later. Reducing conditions persisted up to six months, continuing into subsequent dry periods until the next major oxidizing infiltration event. Evidence of denitrification in shallow groundwater at the site is supported by median nitrate-nitrogen less than 0.016 mg/L, excess nitrogen gas up to 3 mg/L progressively enriched in delta-15N during prolonged basin flooding, and isotopically heavy delta-15N and delta-18O of nitrate (up to 25 and 15 per mil, respectively). Isotopic enrichment of newly infiltrated stormwater suggests denitrification was partially completed within two days. Soil and water chemistry data suggest a biogeochemically active zone exists in the upper 1.4 m of soil, where organic carbon was the likely electron donor supplied by organic matter in soil solids or dissolved in infiltrating stormwater. The cyclic nature of reducing conditions effectively controlled the nitrogen cycle, switching nitrogen fate beneath the basin from nitrate leaching to reduction in the shallow saturated zone. Soil beneath the sandy stormwater infiltration basin was amended using biosorption activated media (BAM) to study the effectiveness of this technology in reducing inputs of nitrogen and phosphorus to groundwater. The functionalized soil amendment BAM consists of a 1.0:1.9:4.1 mixture (by volume) of tire crumb (to increase sorption capacity), silt and clay (to increase soil moisture retention), and sand (to promote sufficient infiltration), which was applied to develop an innovative best management practice (BMP) utilizing nutrient reduction and flood control sub-basins. Construction and materials costs, excluding profit and permit fees, for the innovative BMP were about $US 65 per square meter of basin bottom. Comparison of nitrate/chloride ratios for the shallow groundwater indicate that prior to using BAM, nitrate concentrations were substantially influenced by nitrification or variations in nitrate input. In contrast, for the new basin utilizing BAM, nitrate/chloride ratios indicate minor nitrification and nitrate losses with the exception of one summer sample that indicated a 45% loss. Biogeochemical indicators (denitrifier activity derived from real-time polymerase chain reaction and variations in major ions, nutrients, dissolved and soil gases, and stable isotopes) suggest nitrate losses are primarily attributable to denitrification, whereas dissimilatory nitrate reduction to ammonium and plant uptake are minor processes. Denitrification was likely occurring intermittently in anoxic microsites in the unsaturated zone, which was enhanced by increased soil moisture within the BAM layer and resultant reductions in surface/subsurface oxygen exchange that produced conditions conducive to increased denitrifier activity. Concentrations of total dissolved phosphorus and orthophosphate were reduced by more than 70% in unsaturated zone soil water, with the largest decreases in the BAM layer where sorption was the most likely mechanism for removal. Post-BAM orthophosphate/chloride ratios for shallow groundwater indicate predominantly minor increases and decreases in orthophosphate with the exception of one summer sample that indicated a 50% loss. Differences in nutrient variations between the unsaturated zone and shallow groundwater may be the result of the intensity and duration of nutrient removal processes and mixing ratios with water that had undergone little biogeochemical transformation. In order to quantify potential processes leading to observed nitrogen losses beneath the innovative BMP, an integrated infiltration basinâ€"nitrogen reduction (IBNR) system dynamics model was developed. Based on two simulation periods, the IBNR model indicated denitrification accounted for a loss of about one-third of the total dissolved nitrogen mass inflow and was occurring predominantly in the BAM layer. The IBNR model results in combination with the field-based biogeochemical assessment demonstrated that the innovative BMP using the functionalized soil amendment BAM is a promising passive, economical, stormwater nutrient-treatment technology. Further field- and laboratory-scale research on the long-term sustainability of nutrient losses and further elucidation of causative physicochemical and biogeochemical mechanisms would contribute to improved BAM performance and green infrastructure development in the future.

Research was performed at a pilot-scale wastewater treatment plant operating an adsorption/bio-oxidation (A/B) process at 20C. The study compared B-Stage performance under DO Control, Ammonia Based Aeration Control (ABAC), and Ammonia vs. NOx (AvN) control. AvN in 1) fully-intermittent and 2) intermittently-aerated MLE configurations was compared to DO Control and ABAC, each with continuous aeration, in an MLE configuration. The study also examined operation of each aeration strategy with two different feed types: A-Stage effluent (ASE) and primary clarifier effluent (PCE). Operating modes were compared on the basis of nitrogen removal performance, COD utilization efficiency for denitrification, and alkalinity consumption. AvN was found to provide comparable nitrogen removal performance to DO Control and ABAC. The highest nitrogen removal performance was seen when operating DO Control (81.4 ± 1.2%) and ABAC (81.1 ± 1.2%) with PCE. High nitrogen removal efficiency (77.5 ± 6.1%) was seen when fully-intermittent AvN operation was fed ASE containing a high particulate COD fraction. A high effluent nitrite accumulation ratio (NAR = NO2-/(NO2-+NO3-)) was seen during this period (46 ± 15%) accompanied by the out-selection of Nitrospira. Feeding effluent from AvN control to an Anammox MBBR improved removal efficiency. Increased soluble COD loading resulted in greater nitrogen removal with strategies operating in an MLE configuration while particulate COD was found to be important for processes where removal was designed to occur in downstream reactors. Efficiency of COD for denitrification was found to vary based on the amount and type of influent COD; however AvN in an MLE configuration was found to use COD more efficiently than fully-intermittent AvN. In either configuration, AvN required less alkalinity addition than DO Control or ABAC. High sCOD concentrations in PCE led to increased nutrient removal as compared to ASE but increased heterotrophic growth and mixed liquor concentrations in the B-Stage making the A-Stage an attractive option for its ability to control the C/N ratio fed to BNR processes. / Master of Science

Treatment wetlands play an important role in reducing nitrogen content in wastewater and agricultural run-off water. The main removal process is denitrification and the removal efficiency depends on the hydrological and microbiological features of the wetland, especially in terms of water residence times and denitrification rates. The aim of this thesis was to increase the understanding of the coupled hydrological and microbiological processes regulating the denitrification capacity. This was done by applying a broad spectrum of analyses methods, including tracer experiment, water flow modeling, denitrification rate measurements, and analyses of the microbial community structures. The tracer experiment and flow modeling revealed that the wetland design, especially the vegetation, largely can affect the water residence time distributions in wetlands. In the investigated wetland, vegetation dominated the water flow, explaining 60-80% of the variance in water residence times, whereas basin shape only explained about 10% of the variance, but also mixing phenomena significantly affected the residence times and could considerably delay solutes. Measured potential denitrification rates in the wetland exhibited significant spatial variations, and the variations were best described by concentration of nitrogen in sediments and water residence time. Analyses of the denitrifying bacteria populations indicated that a few key populations dominated and that the community diversity increased with decreasing nutrient levels and increasing water residence times. Moreover, it was found that denitrification rates in terms of Menten and first order kinetics can be evaluated by fitting a mathematical expression, considering denitrification and other nitrogen transforming processes to measured product formation in nitrate limited experiments. / QC 20101110

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