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Since penicillin was discovered in 1928, antibiotic usage in human and veterinary medicine and prevalence of antibiotic resistant bacteria (ARB), has been increasing. While antibiotics and antibiotic resistance genes (ARGs) naturally occur in soils, increasing abundances of ARGs correlate with increased antibiotic usage in agricultural settings. When livestock are treated with antibiotics, the antibiotic compounds, ARB, and ARGs can enter soil via manure excreted onto pastures or applied to other fields as fertilizer, thereby spreading antimicrobial resistance (AMR) in the environment. In addition to human health implications, increased AMR has negative impacts on ecosystem services such as carbon and nitrogen cycling. While many studies have researched antibiotic persistence in agricultural systems and their impacts on soil microbial communities, there are still significant knowledge gaps around the long-term effects of antibiotic exposure in soils, how those impacts differ among soils, and how elements of AMR may differentially transport through soil. To address these knowledge gaps, our objectives were to 1) examine the impact of multi-year repeated additions of manure from cattle administered antibiotics on soil microbial communities, 2) determine the interactive effects of soil moisture and type on soil microbial communities exposed to antibiotics and manure, and 3) differentiate between vertical transport of AMR in the form of viable ARB or ARGs in extracellular plasmids. Our results demonstrate that soil bacterial community structures were consistently altered by 3-year additions of manure from cattle administered antibiotics compared to soil amended with antibiotic-free manure. Furthermore, ARG abundances were higher in soils with manure additions compared to soil without manure, although this was true regardless of whether the cattle were administered antibiotics, suggesting that manure and antibiotic impacts on soil microbial communities can persist over multi-year of repeated manure applications. Additionally, in microcosms, effects of manure from cattle administered antibiotics on ARG abundances, microbial community structures, respiration, and nitrogen pools in soil were seen across multiple soil types and moisture contents, suggesting environmental conditions can alter how manure and antibiotics impact microbial community structure and nutrient cycling. Finally, ARB flowed readily through saturated soil, but were also detectable in the top 5 cm of soil columns. However, ARGs on extracellular plasmids did not flow through soil columns and were not detected in soil, indicating that extracellular DNA does not persist or transport through the soil to any meaningful degree. Overall, these results indicate a nuanced approach is required to mitigate the environmental spread of AMR. Soil management strategies for addressing the AMR crisis should consider the broader context of manure management, as high ARG abundances can come from application of manure from antibiotic-free cattle, and soil microbial communities in individual environments may have varied responses to manure antibiotic exposure. Furthermore, the transport of AMR through soil is complex and dynamic, as elements of AMR may transport differently through soil and require separate consideration in modeling and management. Future AMR management practices that consider diverse factors that affect persistence and spread of AMR in the environment can help protect livestock productivity and maintain the efficacy of antibiotics to protect human and animal health. / Doctor of Philosophy / Antibiotics are an important tool used to fight infections in humans, pets, and livestock. As antibiotics are used more frequently, the bacteria they target are more likely to develop resistance to the antibiotics, leading to increasing cases of infections that are harder to treat and higher risk. Antibiotic resistance can persist and spread in multiple forms, including the antibiotic compounds themselves, as antibiotic resistant bacteria (ARB), or as the genetic material that encodes for antibiotic resistance genes (ARGs). In agricultural systems, when livestock are treated with antibiotics they can excrete the antibiotics, along with ARB and ARGs, in the manure, which is then applied to land as fertilizer. In addition to the associated health risks, the spread of antibiotic resistance impacts microscopic bacteria and fungi in the soil, which are important for recycling nutrients for plants and maintaining ecosystem health. The overall goal of this dissertation was to gain a better understanding of how manure from cattle given antibiotics impacts these bacteria and fungi when manure is applied to the soil. The specific objectives were to 1) look impacts after long-term (multiple years) of manure addition, 2) examine how bacteria and fungi might respond differently to antibiotics in soils of different type or with different amounts of water, and 3) determine if ARGs that exist as free genetic material outside of living bacteria can be moved through the soil with flowing water in the same way as living bacteria. Results showed that while the composition of bacterial and fungal communities in the soil vary from year to year, adding manure with and without antibiotics had both caused different and consistent changes on the composition of bacterial communities. There were also higher concentrations of ARGs in soil that had manure added, however antibiotics in the manure did not cause ARGs to increase further, suggesting that even antibiotic-free manure can impact the spread of antibiotic resistance. Experimental work also demonstrated that the soil type and water content of soil can alter how bacteria and fungi respond to antibiotics in manure. The composition of bacterial and fungal communities, their activity rates, and the amount of nitrogen – an important plant nutrient with availability that is strongly affected by microbial activity – all differed with soil type and water content. Thus, while antibiotic resistance antibiotic resistance can cause measurable changes in soil across a range of environmental conditions, it is also likely to persist and spread in different ways in different environments. Finally, when water containing elements of AMR was added to soil, ARB were shown to both move through the soil easily and remain near the top of soil. In contrast, ARGs contained on genetic material outside of living cells did not move through the soil and were broken down within a few days, suggesting that antibiotic resistance likely spreads through living bacteria more than genes outside of cells. Overall, this work highlights the complexity of understanding the role of environmental transmission in the antibiotic resistance crisis and demonstrates the need for nuanced management approaches that take specific environments and conditions into account.

As climate change exacerbates water scarcity and alters available water and fertilizer resources, it is vital that take appropriate measures to ensure sustainable treatment of water, wastewater, and other waste streams that are protective of public health and support recovery and reuse of water and nutrients. The overarching theme of this dissertation is the advancement of next-generation DNA sequencing (NGS) and computational tools for achieving these goals. A suite of relevant fecal and environmental opportunistic pathogens are examined using both culture-based and NGS-based methods. Of particular concern to this research was not only the attenuation and inactivation of pathogens, but also ensuring that optimal treatment approaches reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Key systems that were the focus of this effort included nutrient reuse (wastewater-derived biosolids and cattle-derived manure), water reuse, and drinking water systems disrupted by a major hurricane. A field study was carried out to survey a suite of pathogens from source-to tap in six small drinking water systems in Puerto Rico six months after Hurricane Maria. The study revealed that pathogenic Leptospira DNA was detected in all systems that were reliant on surface water. On the other hand, Salmonella spp. was detected in surface and groundwater sources and some distribution system waters both by culture and PCR. The study provided comparison of molecular-, microscopic-, and culture-based analysis for pathogen detection and highlighted the need for disaster preparedness for small water systems, including back-up power supply and access to chlorination as soon as possible after a natural disaster. A second field-study examined wastewater derived solids across an international transect of wastewater treatment plants in order to gain insight into the range of ARG concentrations encountered. It was found that, while total ARGs did not vary between treatment or continent of origin, clinically-relevant ARGs (i.e., ARGs encoding resistance to important classes of antibiotics used in humans) were significantly higher in solids derived from Asian wastewater treatment plants. Estimated loading rates of ARGs to soil under a scenario of land application were determined, highlighting in all cases that they are orders of magnitude higher than in the aqueous effluent. Livestock manure, derived from control cattle and cattle undergoing typical antibiotic treatment, and corresponding composts were also evaluated as common soil amendments in a separate study. In this study, the amendments were applied to two soil types in a greenhouse setting, in order to compare the resulting carriage of ARGs on a root (radish) versus leafy (lettuce) vegetable. Remarkably, radishes were found to harbor the highest relative abundance of total ARGs enumerated by metagenomics, even higher than corresponding soils or manures. Although the total microbial load will be lower on a harvested vegetable, the results suggest that the vegetable surface environment can differentially favor the survival of ARBs. The role of wastewater and water reuse treatment processes in reducing ARB and ARGs was also investigated at field-scale. Two independent wastewater treatment plants both substantially reduced total ARG relative and absolute abundance through biological treatment and settling according to metagenomic analysis. The subsequent water reuse treatment train of one system produced water for non- potable purposes and found further reduction in ARGs after chlorination, but a five hundred percent increase in the relative abundance of ARGs in the subsequent distribution system. In the second plant, which employed a membrane-free ozone-biologically-activated carbon-granular activated carbon treatment train for indirect potable reuse, there were notable increases in total ARG relative abundance following ozonation and chlorination. However, these numbers attenuated below background aquifer levels before recharge. Culture-based analysis of these systems targeting resistant ESKAPE pathogens (Escherichia coil, Staphylococcus aureus, Klebsiella spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp.) indicated similar trends as the metagenomic ARG analysis for both systems, but was challenged by sub-optimal media for wastewater samples and low confirmation rates, limiting statistical analysis. In order to advance the application of NGS, molecular, and associated bioinformatic tools for monitoring pathogens and antibiotic resistance in environmental systems, newly emerging methods and field standards for antibiotic resistance assessment were also evaluated. Hybrid assembly, the assembly for both short and long metagenomic sequencing reads, were assessed with an in silico framework in order to determine which available assemblers produced the most accurate and long contigs. Hybrid assembly was found to produce longer and more accurate assemblies at all coverages by reducing error as compared to short read assembly, though the outputs differed in composition from long read assembly. Where it is possible, it is beneficial to sequence using both long- and short-read NGS technologies and employ hybrid assembly, but further validation is recommended. Genome resolved metagenomics has also emerged as a strategy to recover individual bacterial genomes from the mixed metagenomic samples though this is often not well validated. In order to address this, genomes were assembled from reclaimed water systems and were compared against whole-genome sequences of antibiotic resistant E.coli isolates. Metagenome-derived genomes were found to produce similar profiles in wastewater treatment plant influents. A final theme to this dissertation addresses the need to standardize targets, methodologies, and reporting of antibiotic resistance in the environment. A systematic literature review was conducted on assays for the enumeration of key ARGs across aquatic environments and recommendations are summarized for the production of comparable data. In sum, this dissertation advances knowledge about the occurrence of pathogens, ARB, and ARGs across aquatic and agricultural systems and across several countries. Advances are made in the application of NGS tools for environmental monitoring of antibiotic resistance and other targets and a path forward is recommended for continued improvement as both DNA sequencing technologies and computational methodologies continue to rapidly advance. / Doctor of Philosophy / Understanding bacteria in our engineered systems is critical to ensuring drinking water, recycled water, and manure-derived soil amendments are safe for downstream applications. As novel approaches for assessing bacteria are developed, standardized methods and evaluations much be developed to ensure that sound conclusions are made that can appropriately inform policy and practice for the protection of public health. This dissertation focuses on combining bacterial culture and DNA sequencing methods for the study of pathogens (i.e., disease-causing organisms) and antibiotic resistance (i.e., ability of some bacteria to survive antibiotic treatments) in agricultural manure management, water reuse, and drinking water systems. Additionally, this work sought to advance emergent metagenomic analysis tools, which provides a new and potentially powerful pathogen and antibiotic resistance monitoring approach through direct extraction and sequencing of DNA from environmental samples. Antibiotic resistance is a global health challenge and it has been widely recognized that wastewater and agriculture are key control points. When antibiotics are ingested by people or livestock, they select for resistant bacteria in the gut. Mitigation efforts are needed, particularly at wastewater treatment plants and on farms, to ensure that excreted antibiotics and resistant bacteria do not further propagate and pose a risk. However, additional challenges such as climate change have spurred the need for more efficient use of our water and nutrient resources. In this work I examined how nutrient and water reuse treatment methods affect antibiotic resistant bacteria and antibiotic resistance genes using DNA sequencing as well as culture-based methods. In order to assess agricultural practices, a systems approach was conducted at the greenhouse scale to identify key control points to stem the spread of antibiotic resistance when vegetables are grown in soils amended with cattle-derived manure fertilizers. Along the food production chain, vegetables (i.e., radish and lettuce) were found to harbor higher proportions of bacteria carrying antibiotic resistance genes, although the estimated numbers of these bacteria were lower. Solids from an international transect of wastewater treatment plants (Sweden, Switzerland, USA, India, Hong Kong, Phillippenes) were examined because they are also foten used as soil amendments. DNA sequencing of these solids revealed that total measured antibiotic resistance genes did not vary between treatment or continent of origin. Calculations were made to determine the range of total hypothetical outputs of ARGs if the biosolids are land applied. Wastewater reuse systems were also examined using culture and metagenomic DNA analysis so that living pathogens could be compared alongside the total (dead and alive) antibiotic resistance genes. While standard wastewater and subsequent water reuse treatments were found to reduce the absolute numbers of antibiotic resistance genes and bacteria in a treatment plant producing water for non-potable reuse (i.e., irrigation), increases in culturable resistant pathogens and antibiotic resistance genes were apparent in the distribution system (i.e., in the pipes conveying treated water to the point of use). Similar reductions in antibiotic resistant bacteria and resistance genes were also seen in a plant using more advanced treatment (ozonation paired with biofiltration) to produce water suitable for indirect potable reuse via aquifer recharge, but there were indications that ozone and chlorine can increase the proportion of antibiotic resistant bacteria. Finally, genomes were recovered from the metagenomic sequencing analysis and were compared to sequenced culture isolates to validate the capabilities of metagenomic analysis to re-assemble genomes at the strain level, which is often required for pathogen confirmation. Pathogens were also assessed in disrupted drinking water systems in Puerto Rico after Hurricane Maria. Small scale systems that were disrupted by the storm were sampled to identify if pathogens were measurable six months after the hurricane. This work revealed that genes attributed to pathogenic Leptospira were detected in all surface water reliant systems while Salmonella spp. were detected by culture and DNA methods, but only in the source surface and groundwaters, not in the distribution systems delivering water to from the treatment site to the tap. This research also contributed to the advancement of big data analysis pipelines as well as to the standardization of methods to ensure that data produced across studies are comparable. Hybrid assembly, an emergent method that combines both short and long metagenomic DNA sequences generated by different technologies to more accurately recover genomes, was found to improve reliability and accuracy of algorithms aimed at reassembling DNA fragments. Antibiotic resistance is a global challenge, but without standardized methodologies for environmental monitoring, it will be difficult to compare measurements across countries and treatment processes in order to identify effective mitigation strategies. A critical literature review was conducted on assays for the enumeration of key antibiotic resistance genes across aquatic environments so that comparable data can be generated. This will be critical to tap into the tremendous volumes of antibiotic resistance monitoring data being generated around the globe to help identify trends and inform solutions. Collectively, this dissertation advances knowledge about the occurrence of pathogens, antibiotic resistant bacteria and antibiotic resistance genes across aquatic and agricultural systems while also critically evaluating emerging methods for the detection of antibiotic resistance in the environment.

Version 2.0 of the P Index for Virginia uses coefficients describing the risk of P losses for different manure sources applied to fescue pasture that have not been verified on Virginian soils. In the first experiment, four sources of manure (dairy slurry, piggery waste, beef solids, and poultry litter) and triple superphosphate (TSP) were applied iso-nitrogenously to pasture plots (1.5 m2, 10% slope) with 31 ppm Mehlich 1-P soil test. The P treatments were amended in spring at a rate of 62.7 kg P2O5/ha and compared against a no-P-amended control. Forage was cut and removed monthly (n=5). Five rainfall simulations (65-70 mm/h) were conducted at three occasions (June, August, and October); the soil moisture was below field capacity at two events. Continuous surface runoff was collected for 30 min from each plot in accordance with the protocol of the National P Research Project. Data were statistically analyzed using Proc Mixed of SAS with rain event or cutting used as the repeated measure. Runoff concentrations of total P (TP) and dissolved reactive P (DRP) did not vary by treatment. The control showed less TP (0.126 mg/l) and DRP (0.068 mg/l) concentration than all other treatments (ranges 0.190 to 0.249 mg TP/l and 0.129 to 0.182 mg DRP/l) in runoff during the first event (40 d after treatment). The control had the lowest (0.118 mg/l) and TSP the highest (0.248 mg/l) TP concentration during the second event 24 h later. Samples taken at 5-min intervals during the second simulation showed a significant decrease in TP and DRP concentrations over time for all treatments but the control. Treatments did not affect edge-of-the-field losses of TP, DRP, or TKN. Soil test P and water-extractable P measured after the fifth and final rainfall simulation did not correlate to P concentrations in runoff. Forage yields and their N and P concentrations were not impacted. Results indicated a decreasing impact of manure, spring-applied to fescue pasture, on runoff P concentrations throughout the season. Highest TP concentrations were found during the first pair of simulated rainfalls from the TSP treatment. In a second experiment, indoor runoff boxes were used to simulate management intensive rotational grazing. Commercial fertilizer TSP and manure application increased runoff TP concentration from 0.146 mg/l to 0.245 mg/l and DRP concentration from 0.105 mg/l to 0.183 mg/l. Runoff P did not differ between organic or inorganic P treatments, possibly due to the small area of the boxes. However, application of manure increased runoff TKN overall, with a linear decrease as the time increased between application and rain simulation. / Master of Science

Anaerobic digestion and struvite precipitation are two effective ways of treating dairy manure for recovering biogas and phosphorus. Anaerobic digestion of dairy manure is commonly limited by slow fiber degradation, while one of the limitations to struvite precipitation is the availability of orthophosphate. The aim of this work was to study the use of microwave-based thermochemical pretreatment to simultaneously enhance manure anaerobic digestibility (through fiber degradation) and struvite precipitation (through phosphorus solubilization). Microwave heating combined with different chemicals (NaOH, CaO, H₂SO₄, or HCl) enhanced solubilization of manure and degradation of glucan/xylan in dairy manure. However, sulfuric acid-based pretreatment resulted in a low anaerobic digestibility, probably due to the sulfur inhibition and side reactions. The pretreatments released 20-40% soluble phosphorus and 9-14% ammonium. However, CaO-based pretreatment resulted in lower orthophosphate releases and struvite precipitation efficiency as calcium reacts with phosphate to form calcium phosphate. Collectively, microwave heating combined with NaOH or HCl led to a high anaerobic digestibility and phosphorus recovery. Using these two chemicals, the performance of microwave- and conventional-heating in thermochemical pretreatment was further compared. The microwave heating resulted in a better performance in terms of COD solubilization, glucan/xylan reduction, phosphorus solubilization and anaerobic digestibility. Lastly, temperature and heating time used in microwave treatment were optimized. The optimal values of temperature and heating time were 147°C and 25.3 min for methane production, and 135°C and 26 min for orthophosphate release, respectively. Applying manure or slurry directly to the land can contribute to pathogen contamination of land, freshwater and groundwater. Thus it is important to study the fate of pathogens in diary manure anaerobic digestion systems. The goal of the project was to establish a molecular based quantitative method for pathogen identification and quantification, compare the molecular based method with culture based method and study pathogen fate in dairy manure and different anaerobic digesters. Result showed that molecular based method detected more E.coli than the culture based method with less variability. Thermophilic anaerobic digestion can achieve more than 95% pathogen removal rate while mesophilic anaerobic digester had increased E.coli number than fresh manure, indicating temperature is a key factor for pathogen removal. In general, the overall goal of the study is to develop an integrated dairy manure treatment system. The microwave based pretreatment enhanced the subsequent biogas production and struvite precipitation, and the molecular tool based method provided a more precise and faster way to study the pathogen fate in various anaerobic digestions. / Ph. D.

The poultry industry, a vital economic force in Virginia, must dispose of vast amounts of waste, mainly litter (PL) , generated during production processes. This study was conducted to investigate the short-term effects of various rates of PL application, i.e., cumulative 2-year totals of 9.8, 19.5, 29.3, 39.0, and 48.8 mt ha⁻¹, to unimproved pasture. Dry matter yield, N recovery and use efficiency, P recovery and use efficiency, and changes in botanical composition were measured on a mixed species, tall fescue (<i>Festuca arundinacea</i>) and bluegrass (<i>Poa pratensis</i>) pasture. Additionally. the accumulation and movement of P was measured in a Starr clay loam (fme-Ioamy mixed thermic Fluventic Dystrochrepts) by the Mehlich 3 (M3) and Bray 1 (Bl) soil tests. Dry matter yields increased curvilinearly with rate of PL application. This yield increase was attributed to correction of N deficiency in pasture by the PL application. Lower levels of PL increased forage yields in 1992 compared with 1991, due to residual effect of unmineralized N from PL applied in 1991. The PL application rate of 11.4 mt ha⁻¹ in 1991, followed by 8.1 mt ha⁻¹ in 1992 was the most environmentally and economically recommendable rate when compared with inorganic N and P recommended fertilizer rates. Forage yields on this treatment were 21.5 mt ha⁻¹ versus 21. 7 mt ha⁻¹ for the inorganic N and P fertilizer treatment. All rates of applied PL increased the percentage of tall fescue (from approximately 50 percent to > 80 percent) and decreased the percentage of bluegrass in this mixed pasture. Phosphorus from both PL and inorganic sources accumulated in the zone of application. In 1992, the highest rates of PL application had increased M3-extractable P levels by 20 fold over the control (192 mg P kg versus 11 mg P kg⁻¹) and caused movement of P into the 10 to 15 cm soil depth. Levels of P extracted by the M3 and Bl soil tests were highly correlated for both years (r² = 0.96 in 1991 and r² = 0.99 in 1992) but the M3 extracted substantially more P from the surface 0 to 5 cm depth (21 percent in 1991 and 23 percent 1992) while the Bl soil test extracted more P from the 5 to 30 cm soil depth (50 to 66 percent more in 1991 and 20 to 57 percent in 1992). Differences in total acidity and F concentrations account for differences of P extracted by the two procedures. Poultry litter is a suitable source of fertilizer for pasture renovation and production in the Piedmont Region of Virginia. / Master of Science

The objectives of this study were to determine: 1) ammonia emission rates from stored scraped and flushed manure from dairy cows fed either normal or low N diet; and 2) seasonal effects on ammonia emission rates from stored scraped and flushed dairy manure. Four pilot-scale tanks were used for manure storage with different treatments - scraped manure for normal diet (NS), flushed manure for normal diet (NF), scraped manure for low N diet (LS), and flushed manure for low N diet (LF). The first part of the study lasted for 1 month and four treatments were all investigated; the second part of the study lasted for 12 months and two tanks with treatments NS and NF were investigated. Dynamic flux chambers and a photoacoustic gas analyzer were used to measure ammonia emission rates. There was no significant change of the N content of manure as the dietary N content is reduced (from 17.8% to 15.9% crude protein). However, ammonia emission rates from manure storage tanks were reduced by 33% (from 27.4 ± 38.1 to 18.4 ± 21.9 mg m⁻²h⁻¹; P<0.0001 based on paired t-test). Flushing manure reduced emission rates by 72% compared to scraping manure (from 35.6 ± 39.6 to 10.1 ± 8.2 mg m⁻²h⁻¹; P<0.0001 based on paired t-test). Ammonia emission rates for NS, NF, LS and LF were 43.9 ± 48.0, 10.9 ± 8.7, 27.4 ± 27.3, and 9.3 ± 7.8 mg m-2 h-1, respectively. The chamber headspace temperature for NS, NF, LS and LF were 26.0 ± 6.9, 25.8 ± 6.8, 26.6 ± 6.5, and 27.2 ± 6.7 °C, respectively. The manure pH for NS, NF, LS, and LF were 6.3 ± 0.1, 6.4 ± 0.3, 6.4 ± 0.1, and 6.1 ± 0.1, respectively. Both dietary N reduction and manure flushing are recommended to reduce ammonia emission rates from dairy manure storage tanks. Ammonia emission rates were higher in summer and fall, due to higher air temperature and higher manure pH. The pH of scraped manure was 7.2 ± 0.6, 6.7 ± 0.2, 6.5 ± 0.3 and 7.0 ± 0.3 for fall, winter, spring and summer, respectively. The pH of flushed manure was 6.8 ± 0.4, 6.7 ± 0.4, 6.4 ± 0.3 and 6.8 ± 0.4 for fall, winter, spring and summer, respectively. Ammonia emission rates from scraped manure for fall, winter, spring, and summer were 7.4 ± 8.6, -0.5 ± 1.2, 1.1 ± 1.9, and 5.8 ± 2.7 mg m⁻²h⁻¹, respectively. Ammonia emission rates from flushed manure for fall, winter, spring, and summer were 3.9 ± 4.2, -0.5 ± 0.9, 0.8 ± 1.4, and 4.4 ± 1.2 mg m⁻²h⁻¹, respectively. Seasonal changes of air temperature and manure pH were key factors affecting ammonia emissions from manure storage in this study. Seasonal climate conditions including precipitations (rainstorms and snows) and icing can cause reduction of ammonia emissions from manure storage in open air. More attention should be paid to reduce ammonia emissions in warmer seasons, e.g., by covering the storage facilities. / Master of Science

Phosphorus (P) recovery and re-use is very important today for sustainable nutrient cycling and water quality protection due to the declining global P reserves and increasingly stringent wastewater treatment regulations. P recovery as struvite (MgNH4PO4·6H2O) is a promising technology because it can be used as a slow-release fertilizer. The objective of this study was to investigate different pretreatment methods to enhance struvite precipitation in dairy manure. Generally there are two challenges that need to be overcome to precipitate struvite in liquid dairy manure. The first is the relatively high calcium (Ca) concentration. The present study investigated the effectiveness of two calcium binding reagents to reduce the calcium inhibitory effects to enhance the struvite precipitation. A chemical equilibrium model (Visual MINTEQ 2.60) was used to determine the pH to acidify manure and to precipitate struvite. Then, bench-scale experiments were conducted to validate the model results using synthetic and untreated manure. First, the manure was acidified (pH 4.5) to liberate the particulate-bounded Mg2+, Ca2+ and inorganic P. Second, EDTA and/or oxalate compounds were added to the manure. Third, pH was increased to 7.5 for struvite precipitation from the liquid filtrate. Results showed that struvite-containing crystals were obtained from the samples treated with calcium removal compounds, with a total suspended solids (TSS) concentration less than 4,000 mg/L. The second challenge is the low dissolved reactive P (DRP, i.e. PO4-P) to total P (TP) ratio. This study investigated different pretreatment methods for P release to enhance struvite precipitation in liquid dairy manure: enhanced biological phosphorus removal (EBPR), microwave heating (MW) and anaerobic digestion (AD). All of the pretreatment methods resulted in P release but struvite crystals were observed only in precipitates obtained in manure pretreated with MW+H2SO4 acidification. Without oxalic acid to reduce the Ca2+ effects, all the other pretreatment methods were not effective in enhancing struvite precipitation in liquid dairy manure. Hardly any struvite or crystalline-like solids were found in the non-centrifuged samples, regardless of any pretreatment, indicating the effects of suspended solids and organic matter on struvite precipitation. A partial economic analysis was conducted to assess the chemical costs of P recovery from liquid dairy manure with different pretreatment methods; P recovery amounts as struvite were modeled by Visual Minteq 2.61.Three pretreatment conditions (untreated manure + oxalic acid, MW + H2SO4 acidification, and AD) were selected based on the precipitation results. MW + H2SO4 acidification produced the highest struvite quantities among the pretreatment methods to enhance struvite recovery, also with the lowest chemical addition costs. / Master of Science