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The objectives of this research were to: 1) evaluate the accuracy of the Molly cow model predictions of ruminal metabolism and nutrient digestion when simulating dairy and beef cattle diets, 2) advance representations of N recycling between blood and the gut and urinary N excretion in the model, 3) improve the representation of pH and to refit parameters related to ruminal metabolism and nutrient digestion in the model, 4) investigate how ruminal pH affects the microbial community, expression of carbohydrate-active enzyme transcripts (CAZymes), fiber degradation, and short chain fatty acid (SCFA) concentrations. To achieve the first objective, a total of 229 studies (n = 938 treatments) including dairy and beef cattle data, published from 1972 through 2016, were collected from the literature and used to assess the model accuracy and precision based on root mean squared errors (RMSE) and concordance correlation coefficients (CCC). Only slight mean and slope bias were exhibited for ruminal outflow of NDF, starch, lipid, total N, and non-ammonia N, and for fecal output of protein, NDF, lipid, and starch. However, ruminal pH was poorly simulated and contributed to problems in ruminal nutrient degradation and VFA production predictions. To achieve the second objective, representations including ruminal ammonia outflow, intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea N excretion, were added in the model. Total urea entry, gut urea entry, and urinary urea elimination rates collected from 15 published urea kinetics studies were used to derive related parameters. Significant improvements in predictions of variables describing ruminal N metabolism, blood urea metabolism and urinary N secretion were exhibited after the modifications. To achieve the third objective, a dataset assembled from the literature containing 284 peer reviewed studies with 1223 treatment means was used to derive parameter estimates for ruminal metabolism and nutrient digestions. After refitting the parameters, the model is even more robust in representing ruminal nutrient degradation compared to the initial model. Adding ammonia concentration as a driver to the pH equation increased the precision of predicted ruminal pH, and thereby, the precision of predicted VFA concentrations due to an improved representation of pH regulation of VFA production rates. To achieve the fourth objective, six cannulated Holstein heifers with an initial BW of 362 ± 22 kg (mean ± SD) were subjected to 2 treatments in a cross-over design. The treatments were 10 days of intraruminal infusions of both 1) distilled water (Control), and 2) a dilute blend of hydrochloric and phosphoric acids to achieve a pH reduction of 0.5 units (LpH). Statistical analyses indicated 19 bacterial genera and 4 protozoal genera were affected by low ruminal pH. We observed significant correlations between 54 microbes (43 bacterial and 11 protozoal genera) and 25 enzymes, of which 8 key enzymes participated in reactions leading to SCFA production, suggesting that the ruminal microbial community alters fiber catalysis and fermentation in response to altered pH through a shift in carbohydrate-active enzyme transcripts (CAZymes) expression. Overall, after the modifications and reparameterizations, 19.7 to 37.5% of RMSE with essentially no slope bias and minor mean bias were exhibited for of ruminal and fecal outflow of ADF, NDF, fat, and protein, suggesting the model is properly to represent nutrient degradation and digestion in the bovine. Considering ruminal microbes and CAZymes in predicting ruminal volatile fatty acid concentrations could explain more variance of observations. / Ph. D. / The purpose of this research was to improve ruminal nutrient metabolism and nutrient digestion representations in the Molly cow model. First, the model accuracy and precision were assessed using a dataset including 229 studies (n = 938 treatments) conducted with dairy and beef cattle. The model evaluation results indicated the mechanisms encoded in the model relative to ruminal and total tract nutrient digestion are properly represented. However, ruminal pH was very poorly represented in the model with a RMSE of 4.6% and a concordance correlation coefficient (CCC) of 0.0. Although VFA concentrations had negligible mean (2.5% of MSE) and slope (6.8% of MSE) bias, the CCC was 0.28 implying that further modifications with respect to VFA production and absorption are required to improve model precision. As identified by the residual analyses, the representations of N recycling between blood and the gut were improved by considering ruminal ammonia outflow, intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea N excretion in the model. Observations of total urea entry, gut urea entry, and urinary urea elimination rates were collected from 15 published urea kinetics studies were used to derive related parameters. After the modifications, prediction errors for ruminal outflows of total N, microbial N, and non-ammonia non-microbial N were 39.5, 27.8 and 35.9% of the respective observed mean values. Prediction errors of each were approximately 10% units less than the corresponding values before model modifications and fitting due primarily to decreased slope bias. The revised model predicted ruminal ammonia and blood urea concentrations with substantially decreased overall error and reductions in slope and mean bias. After that, ammonia concentration as a driver was added to the pH equation, and a dataset assembled from the literature containing 284 peer reviewed studies with 1223 treatment means was used to derive parameter estimates for ruminal metabolism and nutrient digestions. Refitting the parameters significantly improved the accuracy and precision of the model predictions for ruminal nutrient outflow (ADF, NDF, total N, microbial N, non-ammonia N, and non-ammonia, non-microbial N), ammonia concentrations, and fecal nutrient outflow (protein, ADF, and NDF). Therefore, the improved model can be used to simulate nutrient degradation and digestion in the bovine. Although minor mean and slope bias were observed for ruminal pH and VFA concentrations, the small values for concordance correlations indicated much of the observed variation in these variables remains unexplained. To further explain variance in ruminal metabolism and understand how ruminal pH affects the microbial community, expression of carbohydrate-active enzyme transcripts (CAZymes), fiber degradation, and short chain fatty acid (SCFA) concentrations, six cannulated Holstein heifers with an initial BW of 362 ± 22 kg (mean ± SD) were subjected to 2 treatments in a cross-over design. We observed 19 bacterial genera and 4 protozoal genera were affected by low ruminal pH, and significant correlations between 54 microbes (43 bacterial and 11 protozoal genera) and 25 enzymes, of which 8 key enzymes participated in reactions leading to SCFA production. In summary, after the modifications and reparameterizations, the model is even more robust to represent nutrient degradation and digestion in bovine compared to the initial model. More variance of observations of ruminal volatile fatty acid concentrations could be explained by considering ruminal microbes and CAZymes expressions in further study.

Food choice and preference have been linked to post-ingestive consequences of food consumption. Many ultra-processed foods deliver calories rapidly and are highly rewarding. In literature surrounding substances of abuse, the speed at which a drug reaches the brain affects its abuse potential; this is known as the "rate hypothesis." Here, we test whether the rate hypothesis of addiction may apply to food, specifically whether caloric availability, or the speed at which carbohydrate becomes available for use, contributes to food reward and preference. To do this, we use beverages with novel flavors (conditioned stimulus (CS)) mixed with either a slow metabolizing carbohydrate (maltodextrin and inulin; CS+Slow), a fast-metabolizing carbohydrate (sucrose; CS+Fast), or no carbohydrate (sucralose; CS-). Participants are given each of these drinks 6 times to consume (conditioning period). 2 of these consumption periods occur during in-lab sessions. In one session, blood glucose is measured over one hour post-consumption. In another, we perform indirect calorimetry to assess post-consumption changes in substrate oxidation rates. At the post-testing session, changes in self-reported liking, wanting, and ad libitum intake of each beverage are recorded. Brain response to each flavor cue (without calories) is measured using fMRI at the post-test. We hypothesize the flavor paired with the CS+Fast will be the most liked, wanted, and consumed. We expect greater BOLD (blood oxygenated level dependent) activation to the CS+Fast relative to the CS+Slow and CS- in the nucleus accumbens and hypothalamus. This is an ongoing study and, here, we present our preliminary analysis of the data. / Doctor of Philosophy / People make food choices every day throughout their lives, but why? Research in the past has shown that there are aspects of an individual's life that may be affecting their preferences for foods. One of the aspects investigated in this analysis is metabolism. The way and speed that the body uses carbohydrates plays a large role in how an individual views food options. Here, we test if the speed at which the body is able to use carbohydrates affects their choices of food and if there are any neural components to these food options. To do this, we tested multiple carbohydrates to determine which were the best for comparisons of slow- and fast-metabolizing. These carbohydrate groups were tested against a drink containing no carbohydrates in two metabolic measurements: blood glucose and energy expenditure. We then used a magnetic resonance imaging scan to test brain activity when participants are given small amounts of each drink without carbohydrates. Each carbohydrate condition was paired with a novel flavor so participants wouldn't have a preconceived idea about the caloric load. We found drinks with sucrose, a common household sugar, had the fastest change in metabolic measures. Additionally, areas in the brain related to rewards and learning were activated by flavors associated with sucrose. This leads us to believe that carbohydrates that are quickly used by your body are more rewarding in the brain and may have implications for preferences down the line.

A 0.2-hectare wetland was constructed in the floodplain of Opequon Creek in Northern Virginia as a best management practice (BMP) for stormwater management. The research goals were to 1) determine if wetland hydrology existed and quantify the role of groundwater exchange in the constructed wetland (CW) water budget, 2) estimate wetland hydraulic characteristics during overbank flows, and 3) quantify the event-scale nutrient assimilative capacity of the constructed wetland. CW water table elevations and hydraulic gradients were measured through an array of nested piezometers. During controlled flooding events, stream water was pumped from the creek and amended with nutrients and a conservative tracer in two seasons to determine hydraulic characteristics and nutrient reduction. Samples were collected at the inlet, outlet structure, and at three locations along three transects along the wetland flowpath. Water table elevation monitoring demonstrated that wetland hydrology existed on the site. The mean residence time of the wetland was found to be 100 min for flow-rates of 4.25-5.1 m3/min. Residence time distributions of the high and low marsh features identified a considerable degree of flow dispersion. Manning's n varied between macrotopographic features and was significantly higher in the spring event as compared to the fall event, likely due to the presence of rigid-stem vegetation. Average wetland n was 0.62. Total suspended solid concentrations decreased with increasing residence time during both experiments. Mass reduction of pollutants were 73% total suspended solids (TSS), 54% ammonia-nitrogen (NH3-N), 16% nitrate-N (NO3-N), 16% total nitrogen (TN), 23% orthophosphate-phosphorus (PO4-P), and 37% total P (TP) in the fall, and 69% TSS, 58% NH3-N, 7% NO3-N, 22% TN, 8% PO4-P, and 25% TP in the spring. Linear regression of mass flux over the event hydrograph was used to determine pollutant removal rates between the wetland inlet and outlet. Pollutant removal rates were determined through linear regression of mass flux and were higher in the spring event than in the fall. Dissolved nitrogen species were more rapidly removed than dissolved phosphorus. TSS, TP, and TN removal were greater and faster than dissolved nutrient species, suggesting that physical settling was the dominant removal mechanism for stormwater pollutants. / Ph. D.

Lactating dairy cows convert 25 to 35% of intake N to milk N, and a part of the remaining N ends up in the environment, causing pollution. Dairy cows absorb amino acids available in the small intestine supplied mainly by digestion of microbial protein and ruminally undegraded feed protein (RUP). Ruminally degradable feed protein (RDP) is the major supplier of N for microbial protein synthesis. Most of the excess RDP will be degraded to ammonia and eliminated as urea in urine. Thus, avoiding excess RDP in dairy cattle diets is important in reducing environmental N pollution. The objectives of the work in this dissertation were to test the hypothesis that lactating dairy cows, when fed varying dietary RDP, can maintain feed intake, milk and milk protein yield, ruminal metabolism, passage of nutrients out of the rumen, and N excretion. The first study investigated the effects of decreasing RDP in lactating dairy cow diets on feed intake, milk production and apparent N efficiency. Forty mid-lactation cows (36 Holstein and 4 Jersey × Holstein cross-breds) were fed a diet containing 11.3% of diet dry matter (DM) as RDP for the first 28 d (covariate period). From d 29 to 47 (treatment period) cows were randomly assigned to 1 of 4 diets containing constant RUP (7.1% of DM) but 11.3, 10.1, 8.8, or 7.6% of DM as RDP. Reducing RDP in diets linearly decreased DM intake and tended to decrease milk yield. Milk protein, fat and lactose contents, milk protein yield, body weight, and plasma essential amino acids were unaffected by reduced dietary RDP. However, milk urea-N concentration and milk fat yield decreased linearly with reduced dietary RDP. The apparent efficiency of N utilization for milk N production increased linearly as dietary RDP was reduced. As RDP declined in diets, linear reductions in DM intake and milk production suggested that these cannot be maintained below NRC recommendations of RDP for cows in this study. The aim of the second study was to test the hypothesis that decreasing dietary RDP in lactating dairy cow diets can maintain ruminal metabolism and flow of nutrients out of the rumen and reduce nitrogen excretion. This study was designed as a replicated Latin square with 4 periods of 21 d each. Four treatment diets containing decreasing RDP and constant RUP similar to the first study were used. Three ruminally and duodenally cannulated and 4 ruminally cannulated lactating Holstein cows were randomly assigned to one of the four dietary treatments. A double marker system with Co-EDTA and Yb-labeled forage as markers was used to determine ruminal outflows of nutrients from omasal samples and nutrients reaching the intestine from duodenal samples. Ruminal microbial protein flow was observed using ¹⁵N as an external microbial marker. Feed intake, milk yield, milk composition, and urine and feces output were determined in the last week of each period. Ruminal fluid samples were taken 2 and 4 h after feeding to determine ruminal NH₃-N and volatile fatty acid concentrations. Outflows of nutrients from the rumen were determined by analyzing omasal samples collected over a 24 h feeding cycle in the last week of each period. Reducing dietary RDP decreased protein intakes while DM and fiber intakes were unaffected. Ruminal NH₃-N concentrations linearly declined and peptides and amino acids were unaffected with reduced dietary RDP. A trend for a linear decline in ruminal outflows of microbial N and total N was observed with decreasing dietary RDP. Ruminal volatile fatty acids concentrations were unaltered by feeding treatment diets. Ruminal outflows of DM and acid detergent and neutral detergent fibers were unaffected by treatments. Treatment diets did not have any effect on milk yield and milk composition. However, milk urea-N and milk fat yield decreased linearly with decreasing dietary RDP. Reducing dietary RDP did not affect milk and milk protein yields but did result in greater body protein mobilization. Fecal N output was unaffected however, urine volume and urine N output decreased linearly suggesting reduced environmental N pollution. There was a trend for a linear decrease in total body N balance, but no significant effects on calculated ruminal N balance as dietary RDP decreased. Linear reductions in microbial N leaving the rumen were due to decreased ruminal NH₃-N as peptides plus amino acids and energy supply were unaffected. The linear reduction in milk production and microbial N flow in the first and second studies, respectively, did not support our hypothesis that lactating dairy cows can be fed dietary RDP below current NRC (2001) recommendations without affecting animal performance. The need to raise 15% more cows to alleviate the loss in production may nullify the advantage in reduced N output into the environment by cows fed lower dietary RDP. / 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.

Plants are surrounded by various threats from the environment such as pathogens, abiotic stresses, and animal attacks. Nutrient content and distribution are essential for plant growth and development as well as plant immunity. Pathogens extract nutrients from host plants to benefit their own growth and reproduction. Sulfate, amino acids, and phosphate are indispensable elements for plant growth, plant nutrition, and plant resistance/susceptibility to disease. However, the role of these nutrients in plant-oomycete interactions is an unexplored area. We developed a hydroponic system to precisely control the nutrients applied to plants. We used Arabidopsis thaliana and Nicotiana benthamiana (N. b) as model plants. Hyaloperonospora arabidopsidis as well as two Phytophthora species, Phytophothora capsici (P. cap) and Phytophothora nicotianae (P. nic) were used as model oomycete pathogens. Hpa is an obligate biotrophic pathogen that obtains nutrients directly from the host plant without causing cell death, while P. cap and P. nic are hemibiotrophic pathogens that display a biotrophic phase followed by a necrotrophic phase where they feed on dead cells. Genomic evidence suggests that these pathogens might obtain nutrients including sulfur in different forms from the host (organic and inorganic respectively). We have optimized the hydroponic system and used Taqman PCR assays and sporangiophore counts to assay the influence of sulfur nutrients on Hpa and P. cap infections. We found that (1) sulfur transporter and metabolism genes play essential roles in plant-oomycete interactions; (2) sulfur is critical components for HR responses against Hpa; (3) low sulfur induces pathogenesis related genes as a systemic acquired response. RNA-seq analysis on Phytophthora-infected Arabidopsis suggested that sulfur transport, assimilation, and metabolism play an important role in plant-oomycete interactions. A second project used RNA-seq analysis on P. nic infected N. b, to identify potential nutrition-related-plant genes that are necessary for full pathogen virulence. RNAi knockdowns of N. b AAP6 (amino acid permease 6) and PHT4 (phosphate transporter 4) genes showed an inhibition of oomycete colonization. These experiments together advance the study on the interplay between nutrient assimilation/metabolism in host plants and oomycete infection which will provide insight into the mechanisms how pathogens intercept nutrients from host. In the long-term, this research could reveal new traits applicable for disease resistance to promote crop and food production. / Doctor of Philosophy / Plants are surrounded by diverse threats from the environment such as pathogens, abiotic stresses, and animal attacks. Oomycetes are the most destructive group of pathogens, triggering severe food security issues. Phytophthora is an oomycete genus causing serious economic loss. Traditional disease control managements including pesticides, crop rotation and culture practices, are not time- or financially- efficient due to the difficulty in managing oomycete spread and oomycete resistance to chemicals. Thus, new plant genes for resistance to oomycete diseases would have a major impact. Plant nutrients are critically important for plant fitness in every aspect of plant growth and plant immunity. Cellular regulatory networks for sulfur, amino acids, and phosphate assimilation and metabolism networks connect to every aspect of plant activity such as functioning enzymes, formation of chlorophyll, synthesis of proteins, and plant immunity. These nutrients are part of the plant defense system but also can be beneficial nutrients fed to the invading pathogens. Studying how nutrients are involved in the responses to oomycete invasions will provide information to introduce resistance strategies into crops. We utilized oomycete pathogens with different lifestyles to study the interactions and found that some sulfate transporter genes, an amino acid transporter and a phosphate transporter might be manipulated by oomycete to obtain nutrients. Sufficient nutrition is a critical factor for successfully triggering plant immunity but also could be reprogrammed by pathogens for successful infection and development. Our studies gave useful information to understand which plant nutrient genes are important during plant–oomycete interactions. These findings could be useful in identifying or engineering new plant genes to control plant diseases.

The sediment-water interface is an active biogeochemical zone within streams, where solutes come in contact with mineral surfaces, biota, and reducing conditions. Here, we sought to examine the influence of light, the sediment water interface, and algae on dissolved organic carbon (DOC), nitrogen, and phosphorus within Maple Creek, an agriculturally impacted stream located in Fremont, Nebraska. Simultaneous continuous injection experiments into replicate open- and closed-bottom chambers were used to control the hydrologic residence time. A bromide tracer was injected, and samples were taken for nutrient analysis in the surface and subsurface water at depths up to 8 cm. Dissolved oxygen (DO) and temperature were recorded in order to monitor biotic production. Experiments were conducted over 10 hours, encompassing both light and dark conditions. Results show a strong biotic influence at the sediment-water interface causing nutrient uptake and changes in carbon quality. Changes are especially pronounced during peak photosynthesis hours. The open-bottom mesocosms consistently showed removal of N and P from the surface water to the subsurface. An increase in DOC flux was observed in the open-bottom mesocosms and the organic matter pool exhibited evidence of microbial reduction. The closed-bottom mesocosm showed NH?⁺ increased likely due to photochemical oxidation. These results show the importance of promoting exchange through the subsurface and across the sediment-water interface due to the positive impact it has on nutrient retention. / Master of Science

We investigated variations in resource availability (nitrate and labile organic carbon, LOC) as determinants of denitrification in sediments of streams in the southern Appalachian Mountains, USA. Stream water and sediments were sampled seasonally in two streams of contrasting nitrate availability, Noland Creek (high NO₃-N) and Walker Branch (low NO3-N). Eight additional streams with varying nitrate levels were sampled once during summer. Stream sediments were incubated at ambient stream temperatures, and nitrous oxide accumulation was quantified following acetylene inhibition of nitrous oxide reduction. Denitrification potential was greater in Noland Creek than Walker Branch and was generally greater in sediments from the higher-nitrate streams. In autumn and spring, nitrate and LOC amendments indicated that denitrification potential in Walker Branch sediments was nitrate limited, with temperature having no effect on rates. Denitrification potential in Noland Creek sediments was not limited by nitrate, but temperature had a significant effect. When Noland Creek seasonal data were corrected to a common temperature, no seasonal differences in denitrification potential were detected. Nitrate-N in the 10 surveyed streams ranged from 10 to 549 mg/L, with the highest NO₃-N levels and denitrification rates generally occurring in the higher elevation streams in the GSMNP. We found that nitrate availability, more than LOC availability, controls potential denitrification in these streams. / Master of Science