Nine Virginia soybeans grown in a single location were compared for their antioxidant properties and isoflavone profiles. The extracts were evaluated for their total phenolic contents (TPC), Oxygen Radical Absorbance Capacity (ORAC), and DPPH™ radical scavenging activities. In order to evaluate efficient preparation methods for soybean antioxidants, three Virginia-grown soybeans were extracted using different extraction strategies. The extraction techniques included soxhlet extraction, conventional solvent extraction, and ultrasonic-assisted extraction (UAE) with 5 different common solvent systems including 50% and 80% aqueous acetone, 50 and 70% aqueous ethanol, and 80% aqueous methanol. The TPC in the soybean extracts and isoflavone compositions were significantly different among cultivars. Malonylgenistin was the major isoflavone in all soybean seeds, accounting for 75-83% of the total measured isoflavones. The V01-4937 variety had the highest total isoflavone and malonylgenistin contents, followed by V03-5794. The antioxidant activities of the soybean extracts were also significantly different. Overall, the V01-4937 soybean was the variety that stood out from the other tested Virginia soybeans because it had the highest TPC, ORAC value, and isoflavone contents as well as the second highest DPPH™ scavenging activity. Ultrasonic treatment improved the extraction of soybean phenolics by more than 50% compared to solvent alone. The UAE with 50% aqueous acetone was the most efficient for extraction of phenolic compounds in the soybean seeds. The conventional and UAE with 70% aqueous ethanol extracts had the highest ORAC values, while the soxhlet methanol extracts had the highest DPPH™ radical scavenging activities. Our results suggest that different extraction technologies have a remarkable effect on soybean antioxidant estimation and the UAE is more appropriate for soybean phenolic extraction because it is less time and solvent consuming than the conventional solvent and soxhlet extractions. The V01-4937 soybean with the highest TPC was evaluated for its antioxidant activity and isoflavone contents in an in vitro digestive system. After gastrointestinal digestion, soybean extracts contained higher TPC and ORAC values than cooked soybean (before digestion) but they were relatively low in DPPH™ radical scavenging capacity. The glucosides, daidzin, genistin, and malonylgenistin showed stability during simulated digestion with 83.3 %, 59.4 %, and 10.7 % recovery, respectively. Aglycones, including daidzein and genistein, were recovered at 37 % and 73.7 %, respectively, after in vitro digestion. In this study, daidzin was the most stable and bioaccessible isoflavone determined using the in vitro digestive system. Among the aglycones, genistein was more stable and bioaccessible than daidzein after digestion. In conclusion, soybean antioxidant activities were different among cultivars and efficient extraction for TPC was found using UAE with 50% aqueous acetone. Furthermore, antioxidant activities were stable during digestion and genistein, within aglycones tested, was the most stable and bioaccessible compound following in vitro digestion. This information may provide manufacturers or researchers information required to develop food or nutraceutical products processed for better bioaccessibility of soybean bioactive components. / Ph. D.

Studies were conducted to determine the role of bioflocculation in the activated sludge unit processes. Laboratory and full-scale studies revealed that bioflocculation is important in determining settling, dewatering, effluent and digested sludge properties (activated sludge properties) and may be vital to the function of all processes related to the above properties. In these studies, it was shown that divalent cations such as calcium and magnesium improved activated sludge properties, whereas monovalent cations such as sodium, potassium and ammonium ions were detrimental to these properties. The divalent cations promoted bioflocculation through charge bridging mechanisms with negatively charged biopolymers (mainly protein and polysaccharide). It was found that oxidized iron plays a major role in bioflocculation and determination of activated sludge properties through surface interactions between iron and biopolymers. Oxidized iron was effective in removing colloidal biopolymers from solution in coagulation and conditioning studies. The research included experiments evaluating effects of potassium and ammonium ions on settling and dewatering properties; effects of magnesium on settling properties; effects of sodium, potassium, calcium and magnesium on effluent quality; effect of solids retention time on effluent quality; and evaluation of floc properties during aerobic and thermophilic digestion. A floc model is proposed in which calcium, magnesium and iron are important to bioflocculation and the functionality of aeration tanks, settling tanks, dewatering equipment and aerobic or anaerobic digesters. It is shown that activated sludge floc properties affect wastewater treatment efficiency. / Ph. D.

Aedes aegypti mosquitoes are found globally and also act as the primary vector of Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. The use of insecticides as the main control strategy against diseases transmitted by this mosquito, is increasingly challenged by emerging resistance. Thus, there is a dire need for the development of novel approaches informed by an improved understanding of mosquito biology, to control mosquito populations and, ultimately, disease transmission. Rhythmic biological processes in mosquitoes help optimize resource exploitation by coordinating behaviors and physiology with fluctuating environmental conditions. Such synchronization enables organisms to adjust their physiology, metabolism, and behavior to predictable external cycles. In mosquitoes, circadian rhythmicity has been demonstrated in their biting and oviposition behavior, as well as their locomotor activity. However, little is known regarding how responses to long-range host cues are modulated by the circadian system. Here we show that both antennal sensitivity and olfactory behavior are time-of-day and odor-specific in Ae. aegypti females. Global transcriptomic analysis in whole heads of Ae. aegypti females reveal chemosensory genes differentially expressed throughout the day, providing insight into the molecular mechanisms behind daily variations in olfactory sensitivity and behaviors. We additionally show an odor-induced activation of mosquito behavior. Mosquito locomotion and behavior are also mediated by physiological state, and activity decreases after blood-feeding. Since the central clock components have been shown in other organisms to be redox-sensitive, we explored the role that diet heme plays in mediating behavioral changes following blood ingestion using artificial blood diets. We found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a meal containing hemoglobin, but peripheral period transcription is reduced throughout the course of digestion following ingestion of a protein meal independent of hemoglobin inclusion. Overall, our results show that Ae. aegypti behavioral rhythms mediated by rhythmic gene expression are plastic and susceptible to external host cues and host blood digestion. This work can be leveraged for future studies investigating mosquito host-seeking and blood digestion to identify novel targets for vector control. / Doctor of Philosophy / Female mosquitoes rely on blood-feeding in order to produce eggs, but can unfortunately act as vectors of disease if they transmit pathogens when biting. Insecticides are currently our strongest main tool for controlling mosquito disease vectors such as Aedes aegypti, the yellow fever mosquito. However, increasing cases of insecticide resistance present new challenges in vector control, and new strategies to prevent vector-borne disease are needed. The Ae. aegypti mosquito is found globally and transmits Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. Mosquitoes exhibit rhythms in their gene expression and behaviors such as biting and activity patterns, in order to optimize energy efficiency and coordinate their biology and behaviors with daily fluctuations in the environment. However, it is unknown how their responses to human host odor cues are modulated by their central timekeeping system in the brain. Mosquitoes primarily find a human host via their sense of smell, or olfaction. Odor molecules in the air, emitted by humans, can be detected by mosquitoes' antennae. Here we show that both antennal sensitivity and behavioral responses to odors are time of day and odor-specific in Ae. aegypti females. We quantified gene transcripts in whole heads of Ae. aegypti females as a measure of gene expression, which revealed that genes involved in odor detection are expressed differently throughout the day, providing insight into the molecular mechanisms behind behavioral observations. We also show that mosquito behavior can be activated by odor exposure, and that their behavioral patterns can be influenced for multiple days following exposure. Mosquito behavior is also influenced by blood-feeding, which reduces mosquito activity and flight. Time-keeping genes in the fly brain have been shown to be sensitive to oxidative stress. Blood contains the protein hemoglobin, which can lead to oxidative stress when digested. Using artificial blood diets that allowed us to include or exclude hemoglobin in the meal, we found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a diet containing hemoglobin, but is reduced in the rest of the body throughout the course of digestion following ingestion of a protein meal, whether hemoglobin was included or not. This work can be leveraged for future studies investigating mosquitoes' rhythms in host-seeking and blood digestion to identify new effective targets for vector control.

Various combinations of multi-stage thermophilic and/or mesophilic anaerobic sludge digestion systems were studied to evaluate their solids reduction, odor generation after centrifugal dewatering and indicator organism reduction in comparison to single-stage thermophilic and/or mesophilic anaerobic digestion systems. Pre-aeration of sludge in a thermophilic temperature was also tested followed by single or multi-stage anaerobic digestion systems. It was found that multi stage systems were capable of greater solids removal and placing thermophilic system in multi stage system enhanced indicator organism destruction below EPA Class A biosolids requirement. However, all the digestion systems in the study showed less than 3 log reduction of indicator organism DNA/g solids, which was much smaller than indicator organism reduction measured by standard culturing method. It was also found that the thermophilic anaerobic digestion system could increase organic sulfur-based odors from dewatered biosolids while placing a mesophilic digester reduced odors. It was exclusively observed from sludges containing high sulfate such as ones in this study. A combined anaerobic and aerobic sludge digestion system was also studied to evaluate their solids and nitrogen reduction efficiencies. The aerobic digester was continuously aerated to maintain dissolved oxygen level below 1 ppm and intermittently aerated. It was found that 90 % or more nitrogen removal was possible at the aerobic SRT greater than 3 days and the optimum aeration ratio could be determined. / Ph. D.

Addition of iron to sewer lines for chemical phosphorus removal is widely practiced around the world. However, high dosage of iron may prove detrimental to the anaerobic digestion process and also lead to higher organic sulfur odors and deteriorating biosolids quality. The following research focuses on finding an alternative to the use of iron in wastewater systems by comparing the roles of iron and magnesium on mesophilic anaerobic digestion, the digested effluent characteristics and odors in biosolids. Three anaerobic digesters were operated, one serving as a control with no additives, and the other two having known doses of iron and magnesium added. Comparison of the effluent characteristics revealed an improvement in the overall performance of the magnesium amended digester (in terms of pH, solids and COD reduction, alkalinity and gas production) over the other two reactors, suggesting the benefits of magnesium addition. Both iron and magnesium were found to be effective in achieving high levels of phosphate removals and reducing nuisance odors in dewatered sludge cakes. / Master of Science

Land application of biosolids has been demonstrated to improve nutrient availability (mainly N and P) and improve organic matter in soils, but the effects of biosolids on C sequestration and N cycling in the Mid-Atlantic region is not well understood. The objectives were: 1) to investigate soil C sequestration at sites with a long-term history of biosolids either in repeated application or single large application; 2) to characterize and compare soil C chemistry using advanced 13C nuclear magnetic resonance (NMR) and C (1s) near edge x-ray absorption fine structure (NEXAFS) spectroscopic techniques; and 3) to compare biosolids types and tillage practices on short-term N availability in the Coastal Plain soils. Biosolids led to C accumulation in the soil surface (< 15 cm) after long-time application in both Piedmont and Coastal Plain soils. The C saturation phenomenon occurred in Coastal Plain soils, thus additional soil C accumulation was not achieved by increasing C inputs from biosolids to the Coastal Plain. Soil organic C from profiles in the field sites was not different at depths below the plow layer (15-60 cm). The quantitative NMR analyses concluded that O-alkyl C was the dominant form in the particulate organic matter (POM), followed by aromatic C, alkyl C, COO/N-C=O, aromatic C-O, OCH3 / NCH and ketones and aldehydes. The aliphatic C and aromatic C were enriched but the O-alkyl C was decreased in the biosolids-amended soils. The changes indicated that the biosolids-derived soil C was more decomposed and, thus, more stable than the control. The NEXAFS spectra showed that O-alkyl C was the dominant form in the POM extracted from biosolids-amended soils, followed by aromatic C, alkyl C, carboxylic C and phenolic C groups. These results were similar to those from NMR analysis. The regression and correlation analyses of C functional groups in the POM between NEXAFS and NMR indicated that both techniques had good sensitivity for the characterization of C from biosolids-amended soils. To evaluate short-term biosolids N availability, a three-year field study to investigate the effects of lime-stabilized (LS) and anaerobically digested (AD) biosolids on N availability in a corn-soybean rotation under conventional tillage and no-tillage practices was set up in 2009-2011. Results showed that both LS and AD biosolids increased spring soil nitrate N, plant tissue N at silking, post-season corn stalk nitrate N, grain yield, and soil total N by the end of the growing season. The same factors used to calculate plant available N for incorporated biosolids can be used on biosolids applied to no-till systems in coarse-textured soils. All these results indicated that the application of biosolids affects the long-term quantification and qualification of soil organic C and also improve short-term N availability in the Mid-Atlantic region. / Ph. D.

The primary purpose of this study is to understand the effects of wastewater sludge feed cations on volatile solids destruction, odor control, and dewaterability. The role of influent feed cations and addition of chemical coagulants to sludge were evaluated for those characteristics following anaerobic digestion. Wastewater sludge samples were obtained from seven municipal wastewater treatment plants. Subsequently, batch digestion of the sludges was performed anaerobically in the laboratory with 30 days of SRT. Conditioning with cationic polymer and dewatering simulating a high solids centrifuge were performed after digestion. It was found that volatile solids destruction and volatile organic sulfur compounds generation increased proportionally as iron content in influent increased. However, they decreased as aluminum content in influent increased. It was also found that as iron content in influent increased, higher optimum polymer dose was required for dewatering. On the contrary, increase in aluminum content in influent resulted in decrease in the amount of optimum polymer dose. Direct addition of iron to the digesting sludge can be the most efficient point of addition with respect to volatile solids destruction, odor control, and conditioning of digested sludge. / Master of Science

The objective of this research was to design an alternative wastewater treatment system for turkey processing plants to recover energy and reduce N and P to allowable discharge levels. The objective included: 1. Determine the quantity and quality of biogas produced from the turkey processing wastewater (TPW) and COD reduction efficiency. 2. Design a waste treatment system and validate proof of concept for simultaneous P and N removal with a goal of attaining effluent concentrations of 0.1 mg/L and 4 mg/L, for P and N, respectively. A lab-scale complete mixed anaerobic digester was used for turkey processing wastewater (TPW) digestion and biogas recovery running for 6 months. Along with the anaerobic digester, a two-sludge system called A2N-SBR consisting of an anaerobic-anoxic sequencing batch reactor and an attached growth post-nitrification reactor was added for biological nitrogen and phosphorus removal running for 3 months. Biogas production yields of 778 + 89 mL/gVSadded and 951.30 mL/g COD were obtained through anaerobic digestion. Also, an energy balance was conducted on a pilot scale digester for a turkey processing plant with wastewater production of 2160 m3/d and using a combined heat and power (CHP) enginefor conversion of biogas to heat and electricity. Although the biogas yield achieved in a complete mixed reactor was relatively lower than yields obtained in previous studies using reactors such as UASB, still a complete mixed reactor can be a good choice for biogas recovery from TPW and can be used for codigestion with some specific turkey processing byproducts for biogas recovery. Nitrogen and phosphorus removal in the A2N-SBR system were 47% and 75%, respectively, and during the study the nitrogen and phosphorus removal mean concentration in effluent did not meet the nutrient limits specified in the objectives. Average TP and TN in the effluent were 3.2 mg/L and 137 mg/L, respectively. Throughout the study, the nitrification reactor biofilm was not completely developed. Incomplete nitrification and poor settling might be the reasons that quality obtained in effluent was low. To improve the process condition in A2N-SBR, online monitoring of pH, dissolved oxygen (DO) and oxidation reduction potential (ORP) can help to optimize each stage in the SBR and stages duration can be set based on the results. / 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.

The aim of this study was to examine how manure-derived value-added products via anaerobic digestion impact the environment and economics of dairy farming. An on-farm anaerobic digester (AD) at Virginia dairy was used in this study. The AD performance evaluated for: (i) biogas production (ii) waste stabilization; and (iii) production of organic fertilizer. Locally available organic waste streams were evaluated for co-digestion with dairy manure to increase biomethane production at the on-farm AD. The effective pasteurization temperature and duration to reduce fecal coliform, E. coli, and Salmonella concentrations in the AD effluent to acceptable levels for use as an organic fertilizer were determined. A partial environmental and economic analysis was conducted on the AD system to determine its effects on the environmental-economic sustainability of dairy farming. The results showed that the manure-derived value-added products from the AD improved environmental health and had the potential to improve the economic sustainability of the dairy farm. The AD stabilized the manure adequately and produced 400 KW of electricity, enough to power 230 US homes. Blending manure with locally available organic materials increased volatile fatty acid production, suggesting the potential to increase biomethane yields. Pasteurization at 70°C is sufficient to reduce pathogen indicating organisms to acceptable levels for the manure to be used as an organic fertilizer. The payback periods range from 4.6 to 11.8 years for the AD investment costs and reductions in direct manure methane emissions of 2,436 tonnes CO2e per year. / Master of Science