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<p dir="ltr">Cropland expansion and intensification negatively impact water quality in downstream waterways through increased nutrient loading in the form of agricultural runoff and the loss of natural features that support water storage and nutrient removal. Nowhere is this more evident than the Corn Belt region of the United States where intensive row crop production generates high crop yields but also disproportionately high nutrient export to the Mississippi River. Depressional wetlands were a defining feature of this landscape prior to agricultural land development and are known to retain high levels of nutrients. Thus, protection, restoration, and creation of these wetlands is an increasingly important part of nutrient mitigation strategies in this landscape. Anoxic soils inherent to these wetlands provide the necessary oxidation-reduction (redox) conditions for nitrogen removal via denitrification, but also promote the production of greenhouse gasses including carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O). Denitrification is a key water quality function via conversion of nitrate to N₂ that can also yield high fluxes of N₂O when incomplete. The key environmental factors that govern efficiency of this process and relative differences in nitrogen removal efficiency among wetland types are not well constrained. To address this gap, denitrification rates, greenhouse gas benthic production rates, greenhouse gas emission rates, and environmental conditions were measured in a series of wetland mesocosms with distinct plant species (reed canary grass, rice cutgrass, arrowhead, and an unplanted control) during peak biomass and plant senescence. My results revealed a denitrification stimulation effect from nitrate pulsing that differed significantly by plant type and that responses varied by stage of plant growth. Specifically, I found that labile carbon components resulting from root exudates, decomposition of plant residues and soil organic matter also positively influence denitrification. Additionally, I found that benthic production of CH₄ and CO₂ varies by plant type, as does surface flux for CO₂. Aerenchyma tissue was identified as a controlling factor in greenhouse gas emissions in the mesocosms, and a determining factor in the relationship between benthic production and system flux of greenhouse gasses. My work shows the importance of plant selection to improve design and management practices in agricultural wetlands.</p>

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.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases (CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem (PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies, beskryf. In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH) van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word. Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose, is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n baie vinnige tempo benut. Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat (PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik 'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk. Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die celBACH operon in E. coli toelaat.