Master of Science / Division of Biology / Walter K. Dodds / Understanding the variability of ecological processes across spatial scales is a central issue in ecology, because increasing scale is often associated with increasing complexity. In streams, measurements of biogeochemical fluxes are important for determining ecosystem health and the downstream delivery of nutrients, but are often collected at scales with benthic areas measured in spatial areas from ~10 cm[superscript]2 to ~100 m[superscript]2 (referred to here as patch and reach, respectively), which are smaller than the scale that management decisions are made. Both biotic and abiotic factors will be important when attempting to predict (i.e. scale) biogeochemical rates, but few studies have simultaneously measured rates and their primary drivers at different spatial scales. In the first chapter, I used a conceptual scaling framework to evaluate the ability to additively scale biogeochemical rates by comparing measurements of ecosystem respiration (ER) and gross primary production (GPP) from patch to reach-scales across multiple sites over a two-year period in a prairie stream. Patch-scale measurements with and without fish (biotic factors) and abiotic factors measured simultaneously with metabolic rates suggest that abiotic conditions are stronger drivers of these rates. Patch-scale rates significantly overestimated reach rates for ER and GPP after corrections for habitat heterogeneity, temperature and light, and a variety of stream substrata compartments. I show the importance of determining abiotic and biotic drivers, which can be determined through observational or experimental measurements, when building models for scaling biogeochemical rates. In the second chapter, I further examined patch-scale abiotic and biotic drivers of multiple biogeochemical rates (ER, GPP, and ammonium uptake) using path analyses and data from chapter 2. Total model-explained variance was highest for ER (65% as R[superscript]2) and lowest for GPP and ammonium uptake (38%). Fish removal directly increased ammonium uptake, while all rates were indirectly affected by fish removal through changes in either FBOM and /or algal biomass. Significant paths of abiotic factors varied with each model. Large-scale processes (i.e. climate change and direct anthropogenic disturbances), and local biotic and abiotic drivers should all be considered when attempting to predict stream biogeochemical fluxes at varying spatial scales.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / John M. Tomich / Success for gene therapy clinical protocols depends on the design of safe and efficient gene carriers. Nature had already designed efficient DNA or RNA delivery devices, namely virus particles. However, the risk of insertional mutagenesis has limited their clinical use. Alternatively, safer approaches involving non-viral carriers have been and continue to be developed. While they have been reported to be less efficient than viral vectors, adding genome editing elements to pDNA makes the integration of corrective sequence site specific moving non-viral gene delivery systems closer to clinical applications. Over the last decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize, quite stable and expected to produce minimally immunogenic and inflammatory responses. We recently reported on a new class of Branched Amphiphilic Peptides Capsules (BAPCs) that self-assemble into extremely stable nano-spheres. BAPCs display a uniform size of _20 nm if they are incubated at 4_C and they retain their size at elevated temperatures. In the presence of DNA, they can act as cationic nucleation centers around which DNA winds generating peptide-DNA complexes with a size ranging from 50nm to 100nm. However, if BACPs are not incubated at 4_C, the pattern of interaction with DNA differs. Depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nano-_bers (0.5-1 _M in length) or condense the plasmid into nano-sized structures (100-400nm). Different gene delivery efficiencies are observed for the three types of assemblies. The structure where the DNA wraps around BAPCs display much higher transfection efficiencies in HeLa cells in comparison to the other two morphologies and the commercial lipid reagent Lipofectinr. As a proof of concept, pDNA was delivered in vivo, as a vaccine DNA encoding E7 oncoprotein of HPV-16. It elicited an immune response activating CD8+ T cells and provided anti-tumor protection in a murine model.

Master of Science / Geology / Matthew Kirk / The Cherokee basin in southeastern Kansas is a declining coalbed methane (CBM) field where little is known about how the CBM formed, the extent to which it continues to form, and what factors influence its formation. An understanding of methanogenic processes and geochemistry could lead to potential enhancement of methane formation in the basin. The objectives of this project are to (1) determine the pathway of methane formation and (2) determine whether geochemistry has influenced gas formation. In order to reach the objectives, we analyzed formation water geochemistry, production history, and gas composition and isotopes. Post Rock Energy Corporation gave us access to 16 wells for sampling purposes. We collected gas samples in Isotubes® for compositional and isotopic analyses at a commercial laboratory. We analyzed major ion chemistry from formation water using standard methods. Co-produced water samples we collected are Na-Cl type with total dissolved solids content ranging from 35,367 to 91,565 mg/L. TDS tended to be highest in samples collected from wells with greater total depth. The pH and temperature of sampled water averaged 7.0 and 19°C with an alkalinity ranging from 3.33 to 8.59. Gas dryness and δ¹³C CH[subscript 4] range from 196 to 4531 and -69.95 to -56.5, respectively, which indicate that methane is being produced biologically. Comparing the δ¹³C CH[subscript 4] to the δD CH[subscript 4], which ranges from -228.2 to -217.2, suggest that the primary pathway of methanogenesis is H[subscript 2]/CO[subscript 2] reduction. We calculated Δ (the difference between δ values) in order to correlate isotope data to produced water chemistry. Samples ΔD and Δ¹³C values range from -189.1 to -168.7 and 61.52 to 69.99. Calculated ΔD[subscript CH4-H2O] and Δ¹³C[subscript CO2-CH4] values approach the range for the acetate/methyl pathways as Clˉ concentration increases, potentially indicating a slight shift in methanogenic pathway in deeper, more saline portions of the basin. The culturing results revealed that living methanogens are still able to utilize H[subscript 2], acetate, and methanol present in co-produced formation water from all tested wells.

Master of Science / Department of Agronomy / Charles W. Rice / Biotic interactions among mycorrhizal fungi, nematodes, plants and other microbial communities can have significant effects on the dynamics of C and nutrient cycling. The specific objectives of this study were (1) to evaluate the effects of grazing and mycorrhizal symbiosis on the allocation and storage of C, especially for plant above-and belowground biomass, (2) evaluate the biotic rhizosphere interactions and their role in C cycling, (3) determine the soil microbial community structure as a result of the plant-mycorrhizal symbiosis, and (4) determine the effect of mycorrhizal fungal abundance on soil aggregation. The soil for the experiment was sampled from the Ap horizon of a fine-silty, mixed, superactive, mesic Cumulic Hapludolls located at Konza Prairie Biological Station, Manhattan KS. The experiment was a three-way factorial in a complete randomized block design with four replications. The three factors were mycorrhizae (M), nematodes (N), and phosphorus (P). In a greenhouse study, 96 microcosms (52×32×40cm) were planted to Andropogon gerardii Vit. so that a third of the microcosms could be destructively sampled at the end of each growing season for three years. Plant biomass was separated into aboveground, rhizomes, and roots. All components were dried and weighed at harvest. Mycorrhizal fungi and P increased plant aboveground biomass, while nematodes decreased plant aboveground biomass compared to non-inoculated controls. As expected, P increased plant root biomass, while mycorrhizae increased plant rhizome biomass. Nematodes decreased both above- and belowground biomass. Phospholipid and neutral lipid fatty acid (PLFA and NLFA) analysis were determined for both soil and roots. Water-stable aggregates were separated using a modified Yoder wet-sieving apparatus and analyzed for mass, total C and N, and the isotopic composition of C. There was a positive relationship between AM fungal abundance in the soil and the mass of the largest macroaggregates (>2000µm) after the 3rd year (r=0.67). The effect of roots on the macroaggregate (>2000µm) fraction was not apparent. Phosphorus significantly increased smaller macroaggregates (250-2000µm), along with significantly enhanced plant root biomass, which indirectly demonstrated the effect of roots on the formation of macroaggregates (250-2000µm). The addition of P induced more plant derived C into the aggregates than the non-P amended microcosms as suggested by the [superscript]13C content of the aggregates. Our results confirmed the importance of biotic and abiotic interactions among mycorrhizae, nematodes, and phosphorus on plant growth and the resulting effect on the soil C cycle and soil aggregation.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / Halogenated organic compounds have had widespread and massive applications in industry, agriculture, and private households, for example, as degreasing solvents, flame retardants and in polymer production. They are released to the environment through both anthropogenic and natural sources. The most common chlorinated solvents present as contaminants include tetrachloroethylene (PCE, perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and persistence in the environment, threatening human and environmental health. This contribution provides insight on PCE degradation at laboratory and field scale at a former dry cleaning site in Manhattan, KS. Biostimulation experiments included combinations and concentrations of the following nutrients: soy oil methyl esters (SOME), yeast extract (YE), glucose, lactate, methanol and cheese whey. Bioaugmentation studies used KB-1 bacterial consortium (commercially available culture containing Dehalococcoides). This culture is known to complete the degradation of PCE to a safe end product, ethene. Concentrations of PCE and its degradation intermediates were monitored in the gas phase of the microcosm vials. Biostimulation of the natural ground water and soil microflora did not completely degrade PCE as cis-DCE (c-DCE) accumulated in the sample. Bioaugmented microcosms containing YE and SOME created reducing conditions for KB-1 culture, resulting in ~ 90% dechlorination of PCE to methane and c-DCE. Cheese whey microcosms containing 0.05% cheese whey inhibited the KB-1 culture. This inhibition was due to a drop of pH that inhibited the culture activity. Lower concentrations of cheese whey (e.g. 0.01% to 0.025%) reduced PCE and generated methane in KB-1 augmented microcosms. Based on microcosm results, a pilot bioremediation field study was conducted for a dry cleaning site contaminated with PCE. Ground water flow threatened public water wells located 1.5 miles from the source. Concentrations of PCE in the aquifer was 15 mg/L above the maximum contaminant level of 5 µg/L. Tracer studies with potassium bromide (KBr) were conducted before, during and after the bioremediation study. Nutrient solutions prepared with YE, SOME, lactate and glucose were used for biostimulation and preconditioning of ground water prior to KB-1 injection. Nutrients were provided twice during the pilot study to supplement microbial growth and cheese whey was used. During biostimulation no degradation beyond DCE was evident. The addition of KB-1 reduced PCE and DCE concentrations in the monitoring wells of the pilot study area. Total chlorinated ethene concentrations did not reach background levels 2 years after the last nutrient addition. Tracer results showed that microbial growth decreased ground water velocity during the study, but returned to normal conditions 1 year after the last nutrient addition. In this study we were able to show that native microbial population was not able to degrade PCE to final end products. Therefore, it was necessary to introduce KB-1 culture a long with nutrients to support complete reductive dechlorination of PCE.

Master of Science / Department of Biology / John M. Blair / Anthropogenic activities impact ecosystems in numerous direct and indirect ways, affecting the cycling of carbon (C) and nitrogen (N) on local, regional and global scales. North America tallgrass prairie is an ecosystem profoundly altered by anthropogenic activities, with most native prairie converted to alternate land uses or heavily impacted by other environmental changes. While aboveground responses to anthropogenic drivers have received much attention, the responses of belowground biota, ecological processes, and nutrient allocation to land management and environmental change are poorly documented, especially over long timeframes. This research builds upon a long-term experiment (the Belowground Plot Experiment) initiated in 1986 at Konza Prairie Biological Station (Manhattan, KS). I utilized a subset of treatments to address the effects of annual burning vs. fire suppression and/or chronic N additions on soil C and N dynamics and microbial communities in tallgrass prairie. I measured a suite of soil variables related to C and N cycling during the 2012 growing season, including total soil C and N, microbial biomass C and N, in situ net N mineralization, potential N mineralization, in situ CO2 efflux, and potentially mineralizable soil C. I also assessed changes in microbial community composition using microbial phospholipid fatty acids (PLFA) profiles. Annual burning significantly (p≤0.05) increased the soil C:N ratio and in situ CO2 efflux, while decreasing potential ammonification and nitrification rates. Annual burning also increased total PLFA mass and relative abundance of fungi. Chronic N addition (100 kg N ha-1 year-1) significantly reduced the soil C:N ratio, while increasing total soil N and potential nitrification and ammonification rates. Chronic N addition reduced potential C mineralization, microbial biomass C and N, and altered microbial community composition by increasing abundance of bacterial PLFAs and reducing fungal PLFAs. Sampling date also significantly affected many variables. These results indicate that different fire regimes and chronic N enrichment over decades affects soil C and N pools and transformations, as well as microbial biomass and composition. In total, this study highlights the importance of long-term ecological research and identifies likely changes in tallgrass prairie nutrient dynamics and soil microbial communities under increased N and frequent burning.

Doctor of Philosophy / Department of Agronomy / Gary M. Pierzynski / The bioavailability of Pb and Zn is linked to the solubility of solid phases and other soil chemical characteristics, which is associated with their environmental risk, suggesting that in situ stabilization of these elements can be accomplished by influencing their chemistry. However, more research is needed to investigate the effectiveness of different soil amendments on reducing Pb and Zn bioavailability. A lab study was conducted to evaluate the effects of five different P amendments and time on Pb/Zn speciation in a contaminated soil using synchrotron-based techniques, while a field investigation studied the effects of composted beef manure on plant biomass production and the influence on microbial function, size, and community shifts. In the lab study, the Pb-phosphate mineral plumbogummite was found as an intermediate phase of pyromorphite formation, which has not been documented until now. Additionally, all fluid and granular P sources were able to induce Pb-phosphate formation, but fluid phosphoric acid (PA) was the most effective with time and distance from the treatment. However, acidity from PA increased the prescence of soluble Zn species, which can have negative environmental consequences. Granular phosphate rock (PR) and triple super phosphate (TSP) reacted to generate both Pb- and Zn-phosphates, with TSP being more effective at greater distances than PR. In the field study, compost additions of 269 Mg ha[superscript]1 significantly decreased bioavailable Zn, while increasing estimated available water, plant nutrients, and plant biomass as compared to a contaminated control and low addition of compost (45 Mg ha[superscript]1) over three years. Additionally, compost additions of 269 Mg ha[superscript]1 significantly increased microbial enzyme activities, nitrification, and microbial biomass over the contaminated control through the duration of the study. Increases in microbial activity and biomass are related to increases in total C, available water, and extractable P, while negative relationships were found with electrical conductivity and with bioavailable Zn. The addition of lime or lime plus bentonite with compost did not further reduce metal availability, increase plant biomass, or improve the size or function of microbial communities. High compost additions caused a slight shift in microbial community structure according to phospholipids fatty acid analysis. Increases in the mole percents of both Gram-positive (Gm[superscript]+) and Gram negative (Gm[superscript]-) bacteria were found depending on site. Microbial biomass of Gm[superscript]+, Gm[superscript]-, and fungi were also increased by high compost additions. Results indicate that large additions of compost are needed to increase microbial biomass, improve microbial activity, and re-establish a healthy vegetative community. This study proposes that organic matter and P amendments can be used to stabilize and reduce the bioavailability of heavy metals in soils and mine waste materials, but must be managed carefully and intelligently.

Doctor of Philosophy / Department of Chemical Engineering / Larry E. Erickson / Chlorinated solvents are the second most ubiquitous contaminants, next to petroleum hydrocarbons, and many are carcinogens. Tetrachloroethylene or perchloroethene (PCE) has been employed extensively in the dry cleaning industry and carbon tetrachloride (CT) has been used as a fumigant in grain storage facilities. In this work, remediation feasibility studies were conducted by mesocosm experiments; a chamber was divided into six channels and filled with soil, and plants were grown on top. Each channel was fed with contaminated water near the bottom and collected at the outlet, simulating groundwater flow conditions. The contaminants were introduced starting from March 12, 2004. PCE was introduced at a concentration of about 2 mg/L ([similar to]12 [Mu]moles/L) in three channels, two of them with alfalfa plants and the other with grass. CT was introduced at a concentration of about 2 mg/L ([similar to]13 [Mu]moles/L) in the other three channels, two of them with alfalfa plants and the other with grass. After the system had attained steady state, the concentrations of PCE and CT at inlet and outlet were monitored and the amount of PCE and CT disappearing in the saturated zone was studied. Since no degradation products were found at the outlet after about 100 days, one channel-each for PCE and CT (with alfalfa) was made anaerobic by adding one liter of 0.2 % glucose solution. The glucose solution was fed once every month starting from July 1, 2004 and continued until February 2005. From October 1, 2004, one liter of 0.1 % emulsified soy oil methyl esters (SOME) was fed to two other channels (with alfalfa), one exposed to PCE and another exposed to CT. The SOME addition dates were the same as that for glucose. The outlet liquid of the channel fed with PCE and SOME started to contain some of the degradation compounds of PCE; however, the extent of degradation was not as great as that of the glucose fed channel. No degradation compounds were observed in the outlet solution of the channel (grass grown on top) in which no carbon and energy supplements were added. Similar trend was observed in the CT fed channels also. KB-1, a commercially available microbial culture (a consortium of dehalococcoides) that degrades dichloroethene (DCE), was added through the inlet of the PCE fed channels, but this did not lead to sufficient conversion of DCE. Addition of KB-1 at well 3, located approximately in the middle of the channel, had a greater impact in the degradation of DCE, in both glucose and SOME amended channels, compared to addition at the inlet. KB-1 culture added to the channel was active even 155 days later, suggesting that there is sustainable growth of KB-1 when provided with suitable conditions and substrates. A pilot field study was conducted for remediation of a tetrachloroethylene (PCE) contaminated site at Manhattan, KS. The aquifer in the pilot study area has two distinct zones, termed shallow zone and deep zone, with groundwater velocities of about 0.3 m/day and 0.1 m/day. Prior to the pilot study, PCE concentration in groundwater at the pilot study area was about 15 mg/L (ppm) in the deep zone and 1 mg/L in the shallow zone. Nutrient solution comprising soy oil methyl esters (SOME), lactate, yeast extract and glucose was added in the pilot study area for biostimulation, on August 18, 2005. Potassium bromide (KBr) was added to the nutrient solution as a tracer. PCE was converted to DCE under these conditions. To carry out complete degradation of PCE, KB-1, a consortium of Dehalococcoides, and a second dose of nutrient solution were added on October 13, 2005. After addition of KB-1, both PCE and DCE concentrations decreased. Nutrients were again injected on March 3, 2006 (with KBr) and on August 1, 2006. The total chlorinated ethenes (CEs) have decreased by about 80 % in the pilot study area due to bioremediation. Biodegradation of CEs continued for a long time (several months) after the addition of nutrients. The insoluble SOME may be retained at the feeding area and provide a long time source of electron donors. Biostimulation and bioaugmentation of PCE contaminated soil and groundwater was evaluated in the laboratory and this technique was implemented successfully in the pilot field study. Modeling of the tracer study was performed using an advection-dispersion equation (ADE) and traditional residence time distribution (RTD) methods. The dispersion coefficient, groundwater velocity and hydraulic conductivity were estimated from the experimental data. The groundwater velocities vary from 1.5 cm/d to 10 cm/d in the deep zone and 15 cm/d to 40 cm/d in the shallow zone. The velocities estimated from the 2004 tracer study and 2005 tracer study were higher compared to the velocity estimated from the 2006 tracer study, most likely because of microbial growth and product formation that reduced the hydraulic conductivity. Based on data collected from several wells the hydrologic parameter values obtained from tracer studies appear to vary spatially.

Master of Science / Department of Biology / Walter K. Dodds / Human-induced stressors such as increased nitrogen (N) loadings, altered watershed land-use, and biodiversity losses are a few of the numerous threats to aquatic systems. Prairie streams experience natural disturbances, such as flooding and desiccation, which may alter responses to anthropogenic stressors. Denitrification, the dissimilatory reduction of NO3- to N gas (N2O or N2), is the only permanent form of N removal from terrestrial or aquatic ecosystems, and is important in mitigating N pollution to streams and downstream waters. Little is known about the relationships between denitrification and riparian prairie vegetation or large consumers. In the first chapter, I used outdoor mesocosms to determine the impact of a grazing minnow, Campostoma anomalum, on structural and functional responses of prairie streams to a simulated flood, focusing on denitrification. In terrestrial ecosystems, grazing can stimulate denitrification, but this has not been studied in streams. Ammonium (NH4+) enrichments, used to simulate fish excretion, alleviated N limitations on denitrification. Both fish and NH4+ affected algal biomass accrual, but only fish affected algal filament lengths and particulate organic matter. In a second experiment, I examined the impact of woody vegetation expansion, a primary threat to tallgrass prairie, on riparian and benthic denitrification. Expansion of woody vegetation in these grasslands is due primarily to altered fire regimes, which historically inhibited woody vegetation growth. To determine the effect of woody vegetation expansion on benthic and riparian denitrification, woody vegetation was removed from the riparian zone of a grazed and an ungrazed watershed. Both soil and benthic denitrification rates from this removal buffer were compared to rates in grassy or woody riparian zones. Riparian soil denitrification was highly seasonal, with greatest rates occurring during early spring, and rates being low throughout the remainder of the year. Benthic denitrification was also temporally variable but did not exhibit seasonal trends, suggesting benthic denitrification is driven by factors other than water temperature. Removal of woody vegetation stimulated soil and benthic denitrification rates over rates found in naturally vegetated riparian zones. Elevated N loadings will continue to affect aquatic ecosystems, and these effects may be exacerbated by biodiversity losses or changing riparian vegetation.

Doctor of Philosophy / Department of Agronomy / Ganga M. Hettiarachchi / The milling and mining operations of metal ores are one of the major sources of heavy metal contamination at earth’s surface. Due to historic mining activities conducted in the Tri-State mining district, large area of land covered with mine waste, and soils enriched with lead (Pb), zinc (Zn) and cadmium (Cd) remain void of vegetation influencing ecosystem and human health. It has been hypothesized that if these minewaste materials are disposed of in the flooded subsidence pits; metals can be transformed into their sulfide forms under reduced conditions limiting their mobility, and toxicity. These mine waste materials are high in pH, low in organic carbon (OC) and sulfur (S). The objective of this study was to examine the effect of OC and S addition on the biogeochemical transformations of Pb, Zn and Cd in submerged mine waste containing microcosms. Advanced molecular spectroscopic and microbiological techniques were used to obtain a detail, mechanistic, and molecular scale understanding of the effect of natural and stimulated redox conditions on biogeochemical transformation and dynamics of Pb, Zn and Cd essential for designing effective remediation and mitigation strategies. The results obtained from these column studies indicated that Pb, Zn and Cd were effectively immobilized upon medium (119-day) and long-term (252-day) submergence regardless of treatment. The OC plus S treatment enhanced sulfide formation as supported by scanning electron microscopy- energy dispersive X-ray technique, and synchrotron based bulk-, and micro-X-ray fluorescence and absorption spectroscopy analyses. Microbial community structure changed with OC and S addition with the enhancement sulfur reducing bacteria genes (dsrA/B), and decreased metal resistance genes over time. The long-term submergence of existing mine tailings with OC plus S addition reduced trace metals mobility most likely through dissimilatory sulfate reduction under stimulated reduced conditions. Colloidal assisted metal transportation (<1% of both Pb and Cd) occurred during initial submergence. Retention filters are suggested to avoid colloidal metal transport in order to meet the maximum concentration limit for Pb and Cd in surface and groundwater. This research enhances our understanding of the redox processes associated with the sequestration of non-redox sensitive metals through dissimilatory reduction of sulfates in mine waste materials and/or waste water and provides regulators with useful scientific evidence for optimizing remediation goals.