Soil microorganisms are primarily limited by carbon (C) availability. The majority of C entering belowground food webs comes directly from local flora. Plant derived labile C compounds affect microbial community structure and function, which in turn drive ecosystem function. Research has focused on dissolved organic C (DOC) from litter leachates and root exudates. These compounds are often readily assimilable by soil microorganisms and are precursors for stable soil organic matter formation. Due to diffusion limitation DOC rarely travels far beyond its origin, meaning most soil microorganisms are unable to access these compounds unless they are located near the C source. However, recent studies have illuminated the importance of volatile organic compounds (VOCs) in soil ecosystems. VOCs are produced in abundance and, as vapors, they are able to travel through soil more rapidly than DOC. This dissertation aims to investigate the importance of VOCs commonly produced during the decomposition of leaf litter. We used three separate microcosm experiments to answer the following questions. 1) How do abundant VOCs affect microbial activity in soil? 2) How do VOCs affect nitrogen (N) transformations and the microbes associated with N transformations? 3) How do VOCs affect microbial community composition? 4) Are VOCs from decomposing litter incorporated into soil C pools? In chapter 2, we show that methanol and acetone – common litter derived VOCs – increase microbial activity and labile soil C, while also decreasing available nitrate, and ammonia oxidizing archaea. Interestingly, this decrease in nitrifiers did not affect nitrification rate after VOC addition was ceased. In chapter 3, we demonstrate that soil microbial taxa respond differently to DOC and VOCs at different soil moisture levels. Specifically, DOC primarily affected taxa abundance in wetter soils, while the insoluble VOC α-pinene had the largest impact at lower moisture levels, and methanol affected abundance at all moisture levels. Finally, in chapter 4, we demonstrate that VOCs from decomposing leaf litter altered soil bacterial and fungal communities, and VOC derived C entered all measured soil organic matter pools without direct contact between decomposing litters and the soil. This work demonstrates the importance of VOCs on soil microbial communities and ecosystem function. The VOC induced increase in microbial activity, and the effects of VOCs at low moisture levels suggest that VOCs may function in the bulk soil in a manner similar to DOC in rhizosphere soil. Additionally, the incorporation of VOC-C into soil organic matter pools identifies a hitherto unrecognized mechanism for soil organic matter formation. / Doctor of Philosophy / Soil microorganisms live in an environment where their access to carbon containing compounds limits their growth. In these belowground environments most of the carbon flows from aboveground plant matter through soil microbes into the organisms that consume those microbes. The carbon from plants not only feeds the soil microbes but also changes the type of microbes and how those microbes process important chemicals in the environment – e.g., carbon and nitrogen. Previously, research has focused on carbon compounds that are able to dissolve in water. Often, these compounds originate from liquids that plants release from their roots, or dissolve like tea when leaves are soaked in water. Soil microorganisms can often use these dissolved carbon compounds and directly incorporate them into their biomass. Additionally, these compounds can be stored in soil - sequestering that carbon in the soil, potentially long term. However, dissolved compounds are unable to move very quickly through soil, and the soil microorganisms that live far from the source of these compounds do not have access to them. However, recent studies have found that another form of carbon, volatile organic compounds, are also produced in abundance in the soil environment. These compounds can travel through the air in the soil, as well as in the soil water. When in the air, VOCs travel very quickly and can also travel farther than dissolved compounds. This dissertation aims to investigate the importance of volatile organic compounds that are produced during the decomposition of leaves. We carried out three experiments using small volumes of soil under controlled conditions in the laboratory. We aimed to answer the following questions. 1) How do abundant volatile organic compounds affect microbial activity in soil? 2) How do volatile organic compounds affect microbial processing of nitrogen containing compounds, and the populations of microorganisms that process those compounds? 3) How do volatile organic compounds affect the composition of microorganism in the soil? 4) Are volatile organic compounds from decomposing leaves able to be stabilized in the soil. In chapter 1, we show that methanol and acetone – common volatile compounds produced during the decomposition of leaves– increase microbial activity, and microbial available carbon in soil. Methanol and acetone also decreased available nitrate (an important N containing compound) and a group of organisms that produce nitrate called ammonia oxidizing archaea. Interestingly, once we stopped adding methanol and acetone to the soil the production of nitrate did not differ, meaning that the nitrate producing community was able to recover from the reduction in ammonia oxidizing archaea. In chapter 2, we demonstrated that soil microbial taxa respond differently to dissolved carbon and volatile organic compounds across a gradient of soil moisture. Specifically, dissolved carbon primarily affected taxa abundance in wetter soils, while the insoluble volatile α-pinene had the largest impact at lower moisture levels, and the volatile compound methanol affected abundance of microbial taxa at all moisture levels. Finally, in chapter 3, we demonstrate that volatile organic compounds produced during the decomposition of leaves altered the composition of both bacterial and fungal communities in the soil. Also, and possibly most interestingly, carbon from those volatile organic compounds was stored in all of the pools of carbon that we measured. Together these chapters demonstrate the importance of volatile organic compounds on soil microbial communities and ecosystem function. Since volatile organic compounds induced an increase in microbial activity we are able to infer that soil microorganisms are using these compounds; paired with our observation that volatile organic compounds affected microbial taxa at lower moisture levels than the dissolved compounds did, we can infer that volatile compounds may function as a carbon source in parts of the soil that do not have access to dissolved carbon. Additionally, the incorporation of carbon from volatile organic compounds into soil identified a hitherto unrecognized mechanism for soil carbon sequestration.

Nitrification is the biological process whereby ammonia is converted first to nitrite by ammonia-oxidizing bacteria, and then the nitrite is subsequently converted to nitrate by nitrite-oxidizing bacteria. Ammonia and nitrite levels are closely monitored during treatment of wastewater due to their toxicity to other biological processes. Sybron Chemicals, Inc., is a company that manufactures a nitrifying bioaugmentation culture (1010N) that is used to enhance the naturally occurring levels of biological nitrification. The microbial population of the 1010N product has been examined using a combination of conventional bacteriological methods and modern molecular techniques, with the goal of developing nucleic acid probes that can be used to detect the product in an environmental sample. Small regions of the 16S rRNA genes of the bacteria in 1010N (and two new nitrifying enrichment cultures) were amplified via the polymerase chain reaction (PCR) and analyzed via temperature gradient gel electrophoresis (TGGE). TGGE is a procedure that allows for separation and visualization of individual PCR products that are the same size, based on differences in their sequence. Two of the predominant PCR products in 1010N were purified from the TGGE gel matrix, reamplified via PCR, and sequenced to allow for phylogenetic analysis and nucleic acid probe design. Coincidentally, two strains (NS500-9 and MPN2) that had been isolated from the 1010N mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the 1010N product, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter, respectively, in the existing database. Using the nucleic acid sequences of the cloned DNA, organism-specific DNA probes were designed for both NS500-9 and MPN2. Unfortunately, difficulties were encountered in using the probes to monitor 1010N activity levels via quantitative dot blot hybridizations (rRNA-DNA). Therefore, efforts were redirected to using the TGGE semi-quantitatively with an internal PCR standard (Brüggeman, et al., 2000) to estimate original cell numbers of 1010N within a mixed consortium. This method was not applicable to our system due to substantial preferential binding of the primers to template other than the standard. Samples from a laboratory-scale bioreactor, bioaugmented with 1010N, were used in an attempt to correlate an increase in activity with a detectable shift in population via TGGE. No detectable shift in population was detected in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when 1010N was present in activated sludge. In both whole cell spiking experiments and genomic DNA spiking experiments, it was found that 1010N must be present at a level of at least 5% of the total population in order to be detected. While this provides some information about microbial populations, in order to evaluate the biological activity of a system, nucleic acid probes should be used in a rRNA based study. / Master of Science

Numerous Investigators have reported that NH₄⁺ uptake reduces the yield and quality of flue-cured tobacco (Nicotiana tabacum L.). Metalaxyl, a fungicide commonly used in the production of flue-cured tobacco, has been patented as a nitrification inhibitor. The objectives of this study were to 1) determine the influence of metalaxyl at commonly applied rates on the + nitrification of NH₄⁺ from various sources; 2) study the Influence of soil pH on inhibition of nitrification by metalaxyl; 3) study the Influence of N source and metalaxyl on N accumulation in the plant; and 4) evaluate the influence of N source and metalaxyl on the yield, quality, and chemical composition of flue-cured tobacco leaf. Field, greenhouse, and laboratory experiments were conducted in the Southern Piedmont region of Virginia in 1984 and 1985 to carry out these objectives. Metalaxyl was found to reduce the population of NH₄⁺ oxidizers in soil and inhibit nitrification at applications of 0.56, 1.12, and 3.36 kg ha⁻¹. The inhibitory effects of metalaxyl were much weaker than nitrapyrin. Nitrification was inhibited by metalaxyl only in soils where nitrification was slowed by low pH and wet conditions. In soils of high nitrifying capacity, metalaxyl did not inhibit nitrification. Nitrogen uptake was enhanced by high NO₃⁻ concentrations in the soil, except where metalaxyl reduced NO₃⁻ leaching. Cured leaf concentrations of N were not affected by N source but were slightly reduced by metalaxyl. Nitrogen source and metalaxyl did not affect yield, total alkaloids, or reducing sugars. Quality Index was reduced by decreased soil NO₃⁻ from both N source and metalaxyl. / M.S.

Removal of nitrogen elements in culture water is one of the major concerns in recirculating aquaculture system (RAS). Maintaining a low concentration of nitrogen compounds is essential for a good quality of aquaculture production. Due to fish is very sensitive to the toxic ammonium/ammonia, nitrification biofiltration tank is often an integrate part of filtration in RAS to remove ammonium via nitrification. However, nitrate accumulation via nitrification in RAS is often observed during the operation, which is usually solved by replacing with the fresh water into the system. With the concern of water consumption, bio-electrochemical system (BES) is introduced in this study to realize simultaneous nitrate removal for the system while generating the electricity through electron transferring. A microbial fuel cell (MFC) with an anion exchange membrane (AEM) was constructed. The removal of nitrate from aquaculture water generated from RAS was achieved by nitrate migration across the AEM and heterotrophic denitrification in the anode chamber. To further investigate the potential application of BES in RAS, the cathode chamber was incubated with biofilm to do the nitrification while the denitrification processing in the anode chamber. The study gave a total inorganic nitrogen removal efficiency of 38.72% ± 4.99, and a COD removal of 86.09% ± 9.83. The average daily electricity generation was 67.98 A m-3 ± 13.91, and nitrate-nitrogen concentration remained at 21.02 ± 2.62 mg L-1 throughout the experiment. These results of treating aquaculture water indicate that BES has a potential to install within RAS for enhanced nitrogen removal. / MS

Au Québec, la technologie d’assainissement des eaux usées municipales la plus répandue est le traitement par étangs aérés. L’augmentation des charges et débits envoyés aux étangs ainsi que les exigences de rejet de plus en plus contraignantes forcent la mise à niveau de nombreux étangs. La présente étude porte sur la capacité de dégradation carbonée et de nitrification simultanée du média fixe inerte autoportant immergé BIONESTMD pour augmenter la capacité de traitement des étangs aérés. Des essais ont été réalisés sur douze unités pilotes alimentées en parallèle par un bassin d’égalisation recevant des eaux usées d’origine domestique brutes municipales. Trois charges surfaciques et trois températures d’opération ont été simultanément testées sur les pilotes. Le média a été mis en place dans des cellules flottantes cylindriques. Des taux de dégradation carbonée de plus de 15 g DBO₅Cs/m².d et 25 g DCOs/m².d ont été obtenus. Avec des efficacités de plus de 90% d’enlèvement, des taux supérieurs de dégradation auraient sans doute pu être obtenus à charge plus élevée. Une influence de la température d’opération sur la dégradation de ces deux paramètres a été observée, notamment pour des températures inférieures à 1°C et des charges surfaciques élevées. Les concentrations de DBO₅C, de DCO et de MES mesurées ont montré une dépendance significative en fonction de la charge surfacique et de la température d’opération appliquées. Il a également été observé qu’une température froide et une charge surfacique élevée favorisent le développement d’un biofilm épais. Cette épaisseur favoriserait le détachement du biofilm et la présence de matières particulaires à l’effluent se traduisant par l’augmentation des concentrations de DBO₅C, de DCO et de MES. Des taux de nitrification de plus de 2 g N-NH₄/m².d ont été mesurés pendant les essais et ce malgré des charges surfaciques en matière organique supérieures à la valeur de 5 g DBO/m².d recommandée dans la littérature pour une dégradation carbonée et une nitrification simultanée. Cette capacité du média BIONESTMD, configuré tel que dans cette étude, pourrait être attribuable au biofilm épais favorisant une surface de biofilm et une diffusion des substrats plus élevées que d’autres types de réacteurs à biomasse fixe. Les résultats ont montré une influence importante de la température sur les taux de nitrification, notamment un phénomène de limitation sous 1°C en fonction de la charge en azote ammoniacal. Les coefficients de température mesurés ont montré une dépendance de la charge et de la température. Des conditions limitantes en oxygène accentuent l’influence de la température sur la nitrification. Une équation permettant de déterminer le taux de nitrification à une charge et une température donnée a été développée et utilisée. / In Quebec, the aerated lagoon process is the most popular technology for municipal wastewater treatment. Flowrate and load increases as well as more stringent effluent requirements will make many lagoon upgrades necessary. This study focuses on organic matter removal and simultaneous nitrification of the inert self-supporting immersed BIONESTTM media to upgrade aerated lagoons. Tests were conducted on twelve parallel pilot units fed by an equalisation tank receiving municipal raw domestic wastewater. Three loads and three temperatures were simultaneously tested on the pilots. The media was placed inside cylindrical floating cells. Organic removal rates above 15 g sCBOD₅/m².d and 25 g sCOD/m².d were obtained. With efficiencies above 90% removal, higher organic removal rates would have probably been obtained if the load had been higher. An influence of temperature on the removal of these two parameters was observed, particularly for temperatures below 1°C and high loads. Measured CBOD₅, COD and TSS concentrations showed a load and temperature dependency. It was also observed that cold temperatures as well as high loading rates promote the development of a thick biofilm. This thickness might favor biofilm detachment and particulate matter in the effluent contributing to increased CBOD₅, COD and TSS concentrations. Nitrification rates above 2 g N-NH₄/m².d were measured during the tests despite organic loads higher than the 5 g BOD/m².d recommended in the literature for simultaneous organic matter removal and nitrification. This treatment capacity of the BIONESTTM media, as configured in this study, may be due to the thick biofilm favoring a higher biofilm surface and higher substrate diffusion than other biofilm reactor technologies. Results showed a strong dependency of nitrification on temperature, especially below 1°C where a limitation related to the ammonia load was observed. Measured temperature coefficients showed a dependency on the load and temperature. The influence of temperature on nitrification kinetics was higher in oxygen-limited conditions. An equation allowing the determination of the nitrification rate as a function of the ammonia loading rate and temperature was developed and applied.

M.S.

The objectives of this study were to (1) determine an intermittent aeration strategy that could be used to conserve nitrogen (N) via nitrification in dairy manure, (2) determine the effect of recycled flush water on the bio-availability of N during nitrification, and (3) determine effective and economical dosages of chemicals to remove phosphorus (P) from liquid dairy manure. Intermittent aeration strategies, defined in terms of time the aerator is on and off (ON h:OFF h), could be used to conserve N in dairy manure. Testing of four treatments (continuous aeration [100%], 1h:0.33h [75%], 1h:0.67h [60%], and 1h:1h [50%]) showed that only treatments using air provided for 100% and 75% of the time could support nitrification. The 100% and 75% aeration treatments conserved an average of 38% and 25% of influent total ammonia nitrogen (TAN) as nitrite-N+nitrate-N, respectively. Less than 2% of influent TAN was conserved using 60% and 50% treatments. The effect of manure handling technique on N bioavailability and nitrification was tested using flushed and scraped dairy manure. Nitrification was inhibited in scraped manure. Four aluminum- and iron-based salts and five cationic polyacrylamide polymers were evaluated for P removal using jar tests. Ferric chloride (FeCl3·6H2O), aluminum sulfate (Al2[SO4]3·13H2O, alum), and Superfloc 4512 were selected for further study. Polymer addition enhanced floc size and improved P removal. Treatment of manure (0.89% total solids) from Tank 2 at Virginia Tech's dairy using either FeCl3 or alum in combination with polymer resulted in more than 90% P removal. Chemical treatment and transport of P-rich sludge from a 2,270 cubic meter storage tank would result in an estimated 40% cost savings over transport of the entire manure volume offsite for land application elsewhere. The manure treatment strategies tested provide some solutions to dairy farmers regarding adjustment of N:P ratios so that manure can be applied to meet nutrient needs of crops while adhering to regulations set forth by nutrient management plans. / Master of Science

The purpose of this dissertation was to broadly investigate the fate of antibiotic resistance genes (ARGs) and engineered nanomaterials (ENMs) as representative contaminants of emerging concern in wastewater treatment plants (WWTPs). WWTPs may have their performance impacted by ENMs and may also serve as a reservoir and point of release for both ENMs and ARGs into the environment. Of interest were potential adverse effects of ENMs, such as stimulation of antibiotic resistance in the WWTP, toxicity to microbial communities critical for WWTP performance, and toxicity to humans who may be exposed to effluents or aerosols containing ENMs and their transformation products. Response of nine representative ARGs encoding resistance to sulfonamide, erythromycin and tetracycline to various lab-scale sludge digestion processes were examined, and factors that drove the response of ARGs were discussed. Mesophilic anaerobic digestion significantly reduced sulI, sulII, tet(C), tet(G), and tet(X) with longer solids retention time (SRT) exhibiting a greater extent of removal. Thermophilic anaerobic digesters performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. Bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, was indicated to drive the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition. Effects of silver (nanoAg), zero-valent iron (NZVI), titanium dioxide (nanoTiO2) and cerium dioxide (nanoCeO2) nanomaterials on nitrification function and microbial communities were examined in duplicate lab-scale nitrifying sequencing batch reactors (SBRs), relative to control SBRs received no materials or ionic/bulk analogs. Nitrification function was only inhibited by high load of 20 mg/L Ag+, but not by other nanomaterials or analogs. However, decrease of nitrifier gene abundances and distinct microbial communities were observed in SBRs receiving nanoAg, Ag+, nanoCeO2, and bulkCeO2. There was no apparent effect of nanoTiO2 or NZVI on nitrification, nitrifier gene abundances, or microbial community structure. A large portion of nanoAg remained dispersed in activated sludge and formed Ag-S complexes, while NZVI, nanoTiO2 and nanoCeO2 were mostly aggregated and chemically unmodified. Thus, the nanomaterials appeared to be generally stable in the activated sludge, which may limit their effect on nitrification function or microbial community structure. Considering an aerosol exposure scenario, cytotoxicity and genotoxicity of aqueous effluent and biosolids from SBRs dosed with nanoAg, NZVI, nanoTiO2 and nanoCeO2 to A549 human lung epithelial cells were examined, and the effects were compared relative to outputs from SBRs dosed with ionic/bulk analogs and undosed SBRs, as well as pristine ENMs. Although the pristine nanomaterials showed varying extents of cytotoxicity to A549 cells, and gentoxicity was observed for nanoAg, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed. Studies presented in this dissertation provided new insights in the fate of ARGs in various sludge digestion processes and ENMs in nitrifying activated sludge system in lab-scale reactors. The study also yielded toxicity data of ENMs to biological wastewater treatment microbial communities and human lung cells indicated by a variety of toxicity markers. The results will aid in identifying appropriate management technologies for sludge containing ARGs and will inform microbial and human toxicity assessments of ENMs entering WWTPs. / Ph. D.

As the population in water stressed areas increases, it is critical that wastewater treatment plants (WWTPs) continue to replenish depleted water supplies, and serve as an alternative water source. WWTPs depend on microorganisms in activated sludge to remove pollutants from wastewater and therefore an understanding of how these microorganisms are affected by various conditions and pollutants is needed. Also, as consumer products and industrial processes evolve, so do the pollutants they discharge to wastewater. In order to keep pace with these changes, understanding the effects of emerging contaminants to WWTP processes is essential. The research herein assesses microbial community dynamics of the response of nitrifying microorganisms in activated sludge to variation in ammonia concentration and evaluates the impact of engineered nanoparticles on activated sludge microbial communities and other emerging pollutants, such as antibiotic resistance genes and disinfection by-products. In order to assess microbial community dynamics of the response of nitrifying microorganisms to removal of ammonia in the feed, nitrifying activated sludge reactors were operated at various relevant temperatures and the nitrifying microbial community was characterized using activity assays and bio-molecular techniques. We found that Nitrospira spp. were the dominant nitrifying microorganisms, exhibiting stable relative abundance across multiple trials and over a range of temperatures. These results indicate the possibility of comammox bacteria in the system and highlight the complexity of nitrifying microbial communities in activated sludge relative to past understanding. Both microbial and chemical impacts of engineered nanoparticles on WWTP processes were also investigated. Metagenomic analysis of DNA extracted from activated sludge sequencing batch reactors dosed with gold nanoparticles with varied surface coating and morphology indicated that nanoparticle morphology impacted the microbial community and antibiotic resistance gene content more than surface coating. However, nanoparticle fate was controlled by surface coating more than morphology. Disinfection by-product formation in the presence of nanoparticles during WWTP disinfection was assessed using silver, titanium dioxide, ceria, and zero valent iron nanoparticles. Silver nanoparticles were found to enhance trihalomethane formation, which was attributed to the citrate coating of the nanoparticles. These studies both raise concern over the relationship between engineered nanoparticles and other emerging concerns in WWTPs, and take a step towards informing nanoparticle design in a manner that limits their associated environmental impact. / Ph. D.

Nitrogen (N) fertilizer application constitutes a major portion of farmers' cost of production since N is the most applied nutrient in U.S. cotton production. Despite this, N uptake and use efficiency (NUE) in cotton remains below 50%, which presents challenges of environmental quality. Studies were conducted across 4 states in the US Cotton Belt with the overall objective of evaluating strategies to reduce loss of N to the environment, increase N uptake and NUE. The first study had two objectives: 1) compare NH3 volatilization from surface versus subsurface application/placement of granular (urea) and fluid N source (urea ammonium nitrate; UAN32); and 2) compare NH3 volatilization from urea and UAN treated with enhanced-efficiency fertilizer products. For this study, four A horizon soils of different types were collected from four sites in Virginia (VA), Georgia (GA), Tennessee (TN), and Texas (TX). The EEF products were N-(n-butyl) thiophosphoric triamide (NBPT), nitrapyrin, and ESN. In the first set of experiments (N placement experiments), it was found that across soil types, subsurface placement of granular N source reduced NH3 volatilization by 58 – 81% and subsurface placement of UAN reduced NH3 volatilization by 56 – 98%. In the second set of experiments (EEF experiments), it was found that NBPT reduced NH3 volatilization by 5 – 77% across soil types, and the highest reduction in losses by NBPT was observed on sandier and low CEC soils. Treating urea with both nitrapyrin and NBPT was more effective at reducing NH3 volatilization compared to treating urea with nitrapyrin alone. Based on our findings, subsurface application of granular and fluid N sources is recommended as strategy to reduce NH3 volatilization. Where subsurface placement is not possible, EEF products should be considered. The objectives of the second study were: 1) determine the effects of small grain and legume cover crops on N cycling; 2) evaluate the effects of cover crops and N fertilization on N uptake; and 3) evaluate the effects of cover crops on lint yield. Cover crops were winter fallow (winter weeds), small grain [cereal rye (Secale cereale)], legume mix [(50% crimson clover (Trifolium incarnatum): 50% hairy vetch (Vicia villosa)], and legume mix + rye [(67% legume mix:33% hairy vetch)]. Fertilizer N application rates were 0, 45, 90, and 135 kg ha-1. Soil inorganic N in the top 30 cm depths of the legume mix and legume mix + rye plots was consistently higher than in the rye lone or fallow plots. Cotton lint yield following legume mix with 45 kg ha-1 fertilizer N application was comparable to following fallow plots with 135 kg N ha-1. Thus, fertilizer N rate could considerably be reduced when cotton follows legume cover crops. The objectives of the third study were: 1) evaluate urea and UAN placement (broadcast, dribbling, and injection) on lint yield and fiber quality of three cotton maturity groups (early-, mid-, and full-maturity); (2) assess N use and agronomic efficiencies as influenced by N source, rate, and placement; (3) evaluate the impact of N source and placement on fiber quality. A study including 9 site-years studies was conducted in VA, GA, and TX. It was found that placement had no effect on yield in VA, had effect in all 3 years in TX, and had effect in 1 year in GA. Yield responded to N application in 8 out of 9 site-years in this study. Nitrogen use efficiency was highest among the early- and mid-season varieties. Overall, N rate and variety, rather than application/placement strategy, had the most pronounced effects on lint yield. / Doctor of Philosophy / Nitrogen (N) is usually the major limiting nutrient in cotton production and represents a significant cost of production. On average, the current proportion of applied N recovered in the aboveground crop biomass, (i.e., nitrogen use efficiency, or NUE) ranges from 33 – 50%, meaning that up to two-thirds of applied N is not recovered by crops. This unrecovered N not only represents economic loss to growers, but acts as a potential pollutant in the environment. There is a need for practices which increase N uptake in cotton production, agronomic efficiency, and environmental sustainability. Previous studies conducted outside the U.S. Cotton Belt reported that NUE is influenced by N source and rate of application. Data on NUE of contemporary cotton varieties utilized in the humid and semi-arid regions of the U.S. Cotton Belt would prove useful in efficient N management in the region. First study evaluated gaseous N loss from fertilizer application. It was found that subsurface placement of granular urea reduced NH3 loss as much as 58 – 81% compared to surface broadcast granular urea and subsurface placement of fluid N source reduced NH3 loss by 56 – 98%. In a second study, N rate and method of application/placement were evaluated. Here, it was found that N rate and cotton variety, rather than application/placement strategy had a more pronounced effect on cotton yield. Lastly, the potential of cover crops as alternate N source in cotton production was investigated. It was found that cotton yield following legume mix and fertilized with 45 kg of N per hectare was comparable with cotton yield following no cover crop and fertilized with 135 kg of N per hectare. The results of these studies indicate that subsurface placement of granular and fluid N sources can reduce NH3 loss. In addition, all other things being equal, choosing the right cotton variety as well as applying the right N rate are critical for yield. Furthermore, by growing cotton after legume cover crops, N fertilizer application rates can be significantly reduced.