The hybrid activated sludge-biofilm system called Integrated Fixed Film Activated Sludge (IFAS) has recently become popular for enhanced nitrification and denitrification in aerobic zones because it is an alternative to increasing the volume of treatment plant units to accomplish year round nitrification and nitrogen removal. Biomass is retained on the fixed-film media and remains in the aerobic reactor, thus increasing the effective mean cell resident time (MCRT) of the biomass and providing the temperature sensitive, slow growing nitrifiers a means of staying in the system when they otherwise would washout. While the utilization of media in aerobic zones to enhance nitrification and denitrification has been the subject of several studies and full-scale experiments, the effects and performances of fixed film media integrated into the anoxic zones of biological nutrient removal (BNR) systems have not adequately been evaluated as well as the impacts of integrated media upon enhanced biological phosphorus removal (EBPR). Also, user-friendly software designed specifically to simulate the complex mixture of biological processes that occur in IFAS systems are not available. The purpose of this research was to more fully investigate the effects of integrated fixed film media on EBPR, to evaluate the impacts of media integrated into the anoxic zone on system performance, and to develop a software program that could be used to simulate the effects of integrating the various types of media into suspended growth biological nutrient removal (BNR) systems. The UCT type configuration was chosen for the BNR system, and Accuweb rope-like media was selected for integration into the anoxic zones of two IFAS systems. The media also was integrated into the aerobic reactors of one of the systems for comparison and for further investigation of the performance of the Accuweb media on enhanced nitrification and denitrification in the aerobic zones. The experiments were conducted at 10 day total MCRT during the initial phase, and then at 6 days MCRT for the experimental temperature of 10 oC. A13 hour hydraulic retention time (HRT) was used throughout the study. A high and a low COD/TP ratio were used during the investigation to further study the effects of integrated media on EBPR. The PC Windows based IFAS program began with the concepts of IAWQ model No. 2 and a zero-dimensional biofilm model was developed and added to predict the IFAS processes. Experimental data from the initial study and existing data from similar studies performed at high temperatures (>10oC) indicated that there were no significant differences in BNR performances between IFAS systems with media integrated into the anoxic and aerobic or only aerobic zones and a suspended growth control system maintained at the same relative high MCRT and temperature values. Even though greater biological nitrogen removal could not be achieved for the experimental conditions used, the experimental results indicated that the IFAS systems with fixed film media installed in the anoxic zone have a greater potential for denitrification than conventional BNR systems. As much as 30 percent of the total denitrification was observed to occur in the aerobic zones of the system installed the media only anoxic zones and 37% in the system with integrated media in both anoxic and aerobic zones where as no denitrification was observed in the aerobic zones of the control system when the systems were operated at 6 days MCRT and COD/TP of 52. It is statistically confirmed EBPR can be maintained in IFAS systems as well as Control systems, but the IFAS processes tend to have more phosphorus release in the anoxic zones with integrated fixed film installed. Further, the combination of split flow to the anoxic zone and fixed film media in the anoxic zone resulted in the decreased EBPR performances in the IFAS system relative to the control system. / Ph. D.

Nitrification is increasingly of concern in US potable water systems, due to changes from chlorine to chloramine as a secondary disinfectant in order to comply with new regulations for disinfectant by-products. The ammonia that is released from the chloramine decay supports nitrification. A comprehensive literature review systematically examined the complex inter-relationships between nitrification, materials corrosion and metals release. That analysis suggested that nitrification could accelerate decay of chloramine, enhance corrosion of water distribution system materials, and increase leaching of lead and copper to potable water under at least some circumstances. Moreover, that certain plumbing materials would inhibit nitrification, but that in other situations the plumbing materials would enhance nitrification. Experiments verified that nitrification could affect the relative efficacy of chlorine versus chloramine in controlling heterotrophic bacteria in premise plumbing. Without nitrification, chloramine was always more persistent and effective than chlorine in controlling biofilms. But with nitrification and in pipe materials that are relatively non-reactive with chlorine, chloramine was much less persistent and less effective than chlorine. In materials that are reactive with chlorine such as iron pipes, the relative efficacy of chloramine versus chlorine depends on the relative rate of corrosion and rate of nitrification. High rates of corrosion and low rates of nitrification favor the use of chloramine versus free chlorine in controlling bacteria. Plumbing materials had profound impacts on the incidence of nitrification in homes. Effects were due to toxicity (i.e., release of Cu⁺²), recycling of nitrate back to ammonia substrate by reaction (zero-valent iron, lead or zinc materials), or release of nutrients that are essential to nitrification by leaching from concrete or other materials. As a general rule it was determined that concrete and iron materials encouraged growth of nitrifiers in certain oligotrophic waters, materials such as lead, PVC/plastic pipe, glass and surfaces of other materials were readily colonized by nitrifiers, and materials such as copper and brass were very toxic and relatively resistant to nitrifier colonization. Dependent on circumstance, nitrification had no effect, increased or decreased aspects of materials corrosion. Nitrification markedly increased lead contamination of low alkalinity potable water by reducing the pH. In some cases nitrification dramatically decreased leaching of zinc to potable water from galvanized iron, because of lowered dissolved oxygen and reduced pH. Nitrification did not affect copper solubility in low alkalinity water, but is expected to increase copper solubility in higher alkalinity waters. Finally, nitrification in homes plumbed with PVC or plastics can drop the pH and increase leaching of lead from downstream brass materials in faucets. This can explain why some modern homes plumbed with PVC can have more lead in water when compared to homes plumbed with copper pipe. Phosphate had profound impacts on the incidence of nitrification and resulting effects on water quality. While phosphate levels below about 5 ppb could strongly inhibit nitrification due to a nutrient limitation, nitrifiers can obtain sufficient phosphate from plastic, concrete, copper and iron pipe materials to meet nutritional needs. High levels of phosphate inhibitor can reduce the concentration of Cu⁺² ions and make nitrification more likely, but phosphate can also sometimes lower the corrosion rate and increase the stability of disinfectant and its efficacy in controlling nitrifiers. Phosphate plays a key role in determining where, when and if problems with nitrification will occur in a given water distribution system. This work provides some new fundamental and practical insights to nitrification issues through a comprehensive literature review, lab experiments, solubility modeling and field studies. The results and practical tools developed can be used by utilities and consumers to predict nitrification events and resulting water quality problems, and to make rational decisions about practices such as inhibitor dosing, plumbing material selection and use of whole house filters. / Ph. D.

The goal of my dissertation research was to investigate the structure and function of ammonia-oxidizing microbial communities in temperate forest soils. Accomplishing this goal required a hybrid approach: I used modern molecular biology techniques alongside soil biogeochemical measurements and framed my research using ecological theory largely developed in plant systems. All of my field work was done at Coweeta Hydrologic Laboratory, a Forest Service Station and Long Term Ecological Research Site near Otto, NC. Watershed-level land use manipulations have been performed at Coweeta since the 1930s, including clear-cutting, fertilizing, liming, burning, grazing by cattle, and replanting entire watersheds in white pine. While these treatments were originally imposed to assess the effects of land use on water yield, they have resulted in changes in soil characteristics as well. Working at Coweeta has therefore allowed me to sample ammonia-oxidizer communities across a gradient of soil variables, such as pH and nitrogen (N) availability, within the geographically-constrained area of the Coweeta Basin. First, I used amplicon-based pyrosequencing to independently assess the diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) at several sites within Coweeta. I found that AOA and AOB diversity were a function of both resource availability (i.e. N availability) and environmental harshness (i.e. soil pH) in line with general ecological theory developed for plant systems by Tilman and Grime, respectively. Next, I tested whether AOA and AOB were substrate or nutrient limited in this system by adding either N or a nutrient solution containing both potassium and phosphorus to soil incubations and assessing the growth response of AOA and AOB using quantitative polymerase chain reaction (qPCR). I found strong evidence for substrate limitation by AOB and a marginally-significant positive effect of nutrient addition on growth of AOA. Another intriguing finding from this study was that both AOA and AOB grew during unamended soil incubations. Unamended (buried-bag) incubations have been used to estimate in situ rates of nitrification for over 50 years. By measuring the growth of AOA and AOB alongside nitrification during buried-bag incubations, I discovered that AOA are the dominant ammonia-oxidizers in temperate forest soils. However, I found that AOA are much less efficient at using the energy from ammonia oxidation to create biomass than AOB in the forest soils I sampled. Overall, I found that temperate forest soils contain low abundances of AOA and AOB, with relatively low diversity in both groups. This is especially true for the diversity of AOA, where a single taxon dominated the community at every site. Soil pH and N availability seem to be major selective forces for forest soil ammonia oxidizers, though other nutrients such as potassium and phosphorus may regulate the activity of AOA as well. AOA are most-likely the dominant ammonia oxidizers in temperate forest systems, though this may change with increased disturbance. In a broader sense, I found that ecological theory developed for plant communities was applicable to chemoautotrophic microbes despite the large differences in life history between these groups of organisms. / Ph. D.

Nine recirculating systems at the Virginia Tech Aquaculture Center were placed on line and stocked with yellow perch, <I>Perca flavescens</I>, fingerlings. Fish were stocked at a density of approximately 455 fish m³. Biofilter types were the only factor differing among system designs and were an upflow pulsed bed bead filter, packed tower trickling filter and a rotating biological contactor (RBC). After stocking, systems were allowed to acclimate using ammonia excreted by the yellow perch. Following acclimation, a comparative analysis on biofilter performance began. To evaluate filter performance, water quality parameters tested were temperature (°C), pH, dissolved oxygen (DO), total ammonia-nitrogen (TAN), nitrite-nitrogen (NO₂⁻-N), nitrate-nitrogen (NO₃⁻-N), alkalinity (as CaCO₃), water hardness (as CaCO₃), carbonaceous biochemical oxygen demand (cBOD₃), dissolved organic carbon (DOC), and total suspended solids (TSS). Basic water quality analysis encompassed samples drawn at 8 AM. TAN mass removal analysis encompassed water quality samples drawn at 8 AM and over 24 hours. Higher TAN mass removal rates were achieved in trickling and RBC filters than in bead filters for 8 AM (0.037, 0.14, and 0.004 g/m²/d, respectively) and diurnal sample periods. Analysis of areas under mass removal curves depicted RBC filters as surface area limited. Trickling filters proved most effective at carbon dioxide stripping and pH maintenance and also effectively removed TSS from the culture water. The study did not show filter type as having a significant effect on median organic water quality parameter values. / Master of Science

The research presented in this report is in two sections. Section I involved the performance of a moving bed biofilm reactor (MBBR) versus a biological aerated filtration (BAF) and Section II involved study on causes of deposition in anaerobic digester gas piping. The first section evaluated and compared the performance of a laboratory-scale MBBR and BAF for organic carbon and suspended solids removal. A kinetic study was also performed on the MBBR to evaluate the system performance. The purpose was to recommend one of the systems for the Force Provider project, which provides a containerized "city" for the U.S. Army. The effluent criteria against which the systems were evaluated were total 5-day biochemical oxygen demand (TBOD5) and total suspended solids (TSS) of 30 mg/L each. The report is based on a 5-month laboratory -scale study of the two reactors. The MBBR performance depended on the percent of media provided in the reactor and the organic loading. At a media volume, which displaced the reactor volume by 40 % (heretofore called 40 % media volume), and surface area loading rate (SALR) of 20 g BOD5/m2-d, the system performance deteriorated with time. At 40 % media volume and SALR below 15 g BOD5/m2-d, the system performance improved but still did not meet effluent criteria or average. TBOD5 reduction was generally poor (approximately 50 %). Soluble BOD5 (SBOD5) concentrations were frequently below 30 mg/L and TSS concentrations were often higher than influent TSS. Overall, TSS wastage from the system (both effluent TSS and intentional wastage) averaged 0.032 kg/d. BAF system performance was excellent for TBOD5, CBOD5, SBOD5 and TSS removal, and were consistently less that 30 mg/L. Overall TSS wastage from the BAF (both via effluent and backwash) average 0.027 kg/d and was 16 % less than for the MBBR. Based on demonstrated performance, the BAF was the only viable reactor for the project. Section II of the report focused on possible causes of deposition in an anaerobic digester gas piping at a local wastewater treatment facility (Peppers ferry regional wastewater treatment facility). Industrial waste input to the treatment facility has increased lately and accounts for 40 % of the plant's wastewater inflow. An industry in Pulaski, VA, Magnox Inc. generates and disposes highly concentrated sodium sulfate, (70,000 mg/L) which is a by-product of its activities, to PFRWTF wastewater influent stream. As a result of Magnox industrial waste input, a pilot study was carried out to determine the effect of its waste on the activated sludge treatment units. Results indicated that Magnox industrial waste input would not have adverse effect on the aeration basins. However production of H2S, which can have effect on the anaerobic digester was reported (Olver Inc., 1995). Field analysis of data reported by Olver Inc. (2000) showed that H2S concentration in PFRWTF anaerobic digester gas was rising. X-ray photoelectron spectroscopy analysis of deposits found in the digester pipe together with results obtained from the laboratory-scale study revealed that iron and sulfur played a role in the deposition in the digester gas pipe. The laboratory scale study revealed that ferrous ion in the digester feed possibly precipitated over 90 % of the hydrogen sulfide gas produced in the digester, thus protecting the digester from adverse effects caused by hydrogen sulfide. / Master of Science

A two-stage (carbon oxidation stage one, ammonia oxidation stage two) biological aerated filter was operated for 10 months on-site at a domestic wastewater treatment plant. Over the study, the system was operated at different hydraulic loading rates that resulted in a range of applied organic and ammonia mass loadings. Performance was monitored regularly for water quality parameters in the effluent and along the length of the reactors. It was found that nitrification performance was significantly influenced by organic loading rates greater than 1.2 kg cBOD5/m³-d. Additional experiments were conducted in which a constant mass of ammonia was applied (Phase 1: 1.40 ± 0.08 kg NH₃-N/m³-d; Phase 2: 1.31 ± 0.02 kg NH₃-N/m³-d) to the N column, the second stage of the system, over a range of hydraulic loading rates (5.1 -15.8 m/h). Phases of testing were defined by the background hydraulic loading rate applied to the system (Phase 1: 8.3 m/h; Phase 2: 7.1 m/h) at which the reactors were allowed to reach a steady effluent quality for at least one week prior to testing. Organic loading was minimized and kept relatively constant throughout the hydraulic loading rate experiments (0.65 ± 0.2 kg cBOD5/m³-d) in order to obtain an evaluation of nitrification capacity with minimal competition from heterotrophic bacteria. Results indicated that nitrification performance improved by 17% as the applied velocity increased over the indicated range. A steady-state biofilm model capable of predicting substrate flux was applied to the data in an attempt to explain the improvement in performance with hydraulic loading rate from a fundamental standpoint. Mass transfer coefficients, KL, were derived from the model for conditions in which the experimentally observed flux correlated with the model predictions. Derived KL values were lower than estimations offered by correlation equations but increased with velocity at a similar rate. The model failed to account for changes that may have occurred in biofilm kinetics and structure throughout the length of the reactor. / Master of Science

A pilot-scale, two-stage (carbon oxidation stage one, ammonia oxidation stage two) fixed film biological aerated filter (BAF) process was operated on-site at a domestic wastewater treatment plant. Over the study period, hydraulic loadings to the system were varied, generating a range of organic and ammonia loading conditions. Nitrification was monitored along the length of the filters by measuring chemical nitrogen species and activity levels of ammonia oxidizing bacteria (AOB). During the first phase of the study, nitrification performance was characterized during the wintertime and compared with oligonucleotide probing results using an ammonia-oxidizer specific probe. Overall nitrification efficiency for wintertime conditions (average temperature 12.4 ± 0.1°C) was greater than 90 percent when ammonia-N loadings to the second stage were 0.6 kg/m3-day or less. Nitrification efficiency started to deteriorate at loadings beyond this point. Biofilm and liquid samples were collected along the distance of the two columns at high and low ammonia loadings. The degree of activity observed by ammonia oxidizing bacteria in the biofilm corresponded with the disappearance of ammonia and the generation of nitrate as water passed through the columns. During the second phase of the study, the probing methods were investigated and results of two approaches of analysis were compared to chemical nitrogen profiles. It was found that probe signals normalized to mass of total bacterial nucleic acid corresponded better with chemical profiles than using a novel method of standardizing against known nucleic acid mass standards. During both phases of the study, zones of ammonia oxidizing activity progressed along the length of the columns as organic and ammonia loadings to the system increased. The oligonucleotide probe data suggest that this shift in the location of the nitrifier population is due to higher BOD loads to the second stage, which supported higher levels of heterotrophic growth in the second stage of the system. It was concluded that the strongest influence on nitrification performance in this type of BAF system is likely to be competition between heterotrophs and autotrophs. / Master of Science

Two laboratory experiments were conducted to study the effects of salt and nitrogen-sulfur compounds on the transformations of nitrogen in three Arizona soils. In the first experiment the effect of NaCl in concentrations of 0 to 1 m (molal) at moisture levels of 1/3 and 15 bars was studied in the Gila and Laveen loam soils. At 1/3 moisture nitrification of urea-¹⁵N and native soil nitrogen was appreciably reduced only at 1 m salt level. At 15 bars moisture, nitrification was almost completely inhibited by the 1 m salt concentration. Mineralization of soil nitrogen was reduced more by decreasing moisture than by increasing salt concentrations. Ammonia volatilization was increased by both salt and moisture stress and was associated with inhibition of nitrification. Slight effects of salt were observed on ¹⁵N immobilization and ¹⁵N recovery (including volatilization). In the second experiment nitrogen-sulphur combinations (¹⁵N labelled) of KNO₃, KNO₃ + S, urea, urea + S, APS (ammonia polysulfide) and Thiosul (ammonium thiosulfate) were studied at field capacity (FC) and 1.5 FC moistures. In the calcareous Gila soil nitrification was suppressed by the presence of sulfur at 1.5 FC moisture. Volatilization losses were appreciable only from APS. Immobilization of ¹⁵N was greatest from treatments with the higher sulfur rate (elemental S). Denitrification was slightly increased by sulfur at FC, however, at 1.5 FC dramatic losses occurred by denitrification (autotrophic in the presence of sulfur, especially with elemental S. The nitrifying ability of the slightly acid and coarse textured Sonoita soil was low. Nitrification was suppressed more by the presence of sulfur at both moistures. Ammonia volatilization was appreciable from APS followed by urea. ¹⁵N immobilization was high from urea followed by APS. Appreciable losses by denitrification occurred only with APS. The Sonoita soil showed a lower sulfur oxidizing power than the Gila with the only appreciable rate of oxidation from Thiosul followed by APS.

L’objectif de ce travail est de caractériser l’effet de l’érythromycine sur l’activité bactérienne et l’inhibition de l’épuration dans les réacteurs de traitement des eaux usées urbaines. L’étude a montré l’importance du temps d’exposition à l’érythromycine. L’inhibition de la nitrification et de l’épuration de la DCO a été mesurée sur une période de 4 h pour des concentrations supérieures à 1 mg/L d’érythromycine alors que l’inhibition de l’épuration pour une concentration d’érythromycine de 4 µg/L a été mesurée suite à 20 h d’exposition. L’origine de la liqueur mixte est également déterminante : l’érythromycine a inhibé la nitrification avec les boues de Nancy mais n’a pas inhibé la nitrification avec les boues et Épinal. Par contre, l’inhibition de l’épuration de la DCO a été mesurée pour les deux boues. Des analyses microscopiques ont montré qu’en présence d’érythromycine les flocs bactériens se fractionnaient suite aux lyses bactériennes. La microscopie en épifluorescence et la microscopie confocale combinées à des marqueurs de viabilité fluorescents ont permis de déterminer les vitesses de mortalité bactérienne. Ces études ont mis en évidence un temps de latence qui précède la mortalité des bactéries. Ce temps de latence pourrait être lié aux mécanismes d’adsorption de l’antibiotique et de diffusion dans les flocs, ainsi qu’à la vitesse biologique de mortalité. Enfin, un marquage simultané Gram et viabilité (microscopie confocale 3D au cours du temps) a montré que l’érythromycine pourrait modifier la composition bactérienne des boues en sélectionnant les bactéries les moins sensibles / This study examines the effect of erythromycin on activated sludge bacteria and the inhibition of the pollution removal in batch reactors treating urban wastewater. Results showed the importance of exposure time to erythromycin. Inhibition of nitrification and COD removal was measured during a 4 h period for erythromytcin concentrations higher than 1 mg/L. A 4 µg/L erythromycin concentration inhibited COD removal during a 20 h exposure time. The effect of erythromycin on nitrification was variable depending on the sludge origin. Erythromycin inhibited the specific nitrification rate with sludge from Nancy WWTP, but increased the nitrification rate at the other facility (Epinal WWTP). The cell lysis resulted in destruction of activated sludge flocs. Microscopic techniques (epifluorescence and confocal laser scanning microscopy (CLSM)), combined with a fluorescent viability indicator, allowed us to study erythromycin time-kill activity.Viability staining results showed a latency time before the lower antibiotic concentrations began to kill bacteria. This latency time could be related to antibiotic adsorption and diffusion into activated sludge flocs as well as the rate of bateria death. The effect of erythromycin according to the bacterial Gram type was investigated with 3-dimensional Confocal Laser Scanning Microscopy (CLSM) time-lapse imaging combined with a Gram and Viability staining. Gram+ bacteria had a higher mortality rate than the Gram- bacteria. This result suggests that antibiotic in wastewater could change the activated sludge bacteria composition according to their Gram type by selecting bacteria the less sensitive to the antibiotics

Les Archaea planctoniques contribuent de façon significative aux grands cycles biogéochimiques dans les écosystèmes aquatiques, néanmoins la structure des communautés actives ainsi que leurs variations saisonnières sont encore largement méconnues. En outre, la découverte de l’implication des Archaea dans le cycle de l’azote (Ammonia Oxidizing Archaea ou AOA), plus particulièrement dans le processus de nitrification a considérablement modifié la perception d’un processus autotrophe réalisé uniquement par des bactéries (Ammonia Oxidizing Bacteria ou AOB). Dans les écosystèmes marins, la large distribution des AOA suggère que ces microorganismes joueraient un rôle prépondérant dans le cycle de l’azote néanmoins, ces observations ne sont pas généralisables à l’ensemble des écosystèmes aquatiques en raison de leur grande diversité et/ou d'un manque d'informations et d’études sur certains d'entre eux. Ainsi, les objectifs de ce projet étaient i) d’étudier la structure spatiale et temporelle des communautés d’Archaea actives dans des écosystèmes aquatiques contrastés en termes d’apports anthropiques et/ou de gradients de salinité (lac, estuaire, milieu côtier) ; ii) de déterminer la contribution relative des Archaea au processus d’oxydation de l’ammonium, en comparaison avec celle des bactéries ; et iii) de mieux comprendre les paramètres environnementaux qui pourraient déterminer l’établissement des communautés d’AOA ou d’AOB. / Aquatic Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, especially nitrogen, but details regarding their community structure and seasonal activity and dynamics remain largely unexplored. In marine ecosystems, the widespread distribution of Ammonia Oxidizing Archaea (AOA) suggests that they probably play a major role in nutrients cycling. However, we cannot generalize these observations to all aquatic ecosystems because of their high diversity and/or a lack of information and studies on these organisms for some of these ecosystems. More precisely, lacustrine and coastal ecosystems were less studied while they are potentially subjected to strong anthropogenic impacts. Moreover, notable differences in terms of diversity and activity between marine and freshwater communities can be expected, considering the specific environmental parameters of each ecosystem. The objectives of this thesis were: i) to study the archaeal community structure across a temporal scale and assess the diversity of archaeal communities and AOA in diverse aquatic ecosystems along anthropogenic and/or salinity gradient (lacustrine, estuarine and coastal ecosystems); ii) to determine their relative contribution in ammonia oxidation, compared to Ammonia Oxidizing Bacteria (AOB) by looking at their spatial and temporal distribution and activity, and iii) to explore more precisely the environmental parameters that could drive AOA and/or AOB establishment.