Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Metabolic Flux Analysis for Metabolic Engineering of Marine Organisms(2018) Quinn, Andrew Higgins; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We explored the metabolic pathways in two industrially relevant marine microorganisms to understand their core metabolic capabilities. It is necessary to track how an organism distributes organic building blocks throughout its metabolic pathways so that we can devise strategies to alter its metabolism and reroute substantial metabolic flux towards target compound(s). Though we cannot measure intracellular metabolic fluxes directly, we can retro-biosynthetically calculate them by supplying substrates labeled with non-radioactive isotopes to an organism. We then measure the resulting isotope labeling patterns of metabolites and calculate the fluxes that produced them. We addressed three goals with our research, (i) resolving questions surrounding organic carbon metabolism in the diatom Phaeodactylum tricornutum (P. tricornutum), (ii) identifying reactions in a putative photosynthetic carbon concentrating mechanism in P. tricornutum and (iii) mapping central carbon metabolism of the cellulolytic aerobe Saccharophagus degradans (S. degradans). Towards goal (i) we show that P. tricornutum predominantly consumes glucose, as opposed to atmospheric CO2, under mixotrophic conditions using the Entner-Doudoroff (ED) glycolytic pathway instead of the more common Embden-Meyerhof-Parnas pathway (EMP). We utilized metabolic flux analysis (MFA) to discover that acetate is metabolized for energy production instead of for biomass formation during mixotrophic growth on CO2 and acetate. Finally, we developed a method for measuring isotopic labeling in polyunsaturated fatty acids via gas chromatography-mass spectrometry (GC-MS), and demonstrated its utility in resolving outstanding questions about glucose metabolism by P. tricornutum. Towards goal (ii) we utilized isotope labeling and gene silencing in combination to identify pyruvate carboxylase as a key enzyme in a C4 carbon concentrating mechanism in P. tricornutum, while also ruling out phosphoenolpyruvate carboxylase as a key enzyme in the pathway. Towards goal (iii) we present 13C-MFA of aerobic consumption of glucose, xylose, and cellobiose by S. degradans. This is the first reported MFA of cellobiose metabolism and one of only a handful analyzing xylose metabolism in an aerobic microorganism.Item Processing of Cellulose for the Advancement of Biofuels(2011) Watson, Brian James; Lloyd, Isabel; Hutcheson, Steven; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The enzymatic degradation of cellulose polymers is currently a rate-limiting step in the bioconversion of biomass to biofuels. Cellulose polymers self assemble to form crystalline structures stabilized by a complex network of intermolecular interactions such as hydrogen bonding. The network of interactions in crystalline cellulose (cellulose nanostructure) poses an energy barrier that limits enzymatic degradation as apparent from the activity of Cel5H. To improve the degradability of cellulose the intermolecular interactions must be disrupted. The interactions of the cellulose nanostructure prevent solubilization by water and most other common solvents, but some organic solvents aid degradation of cellulose suggesting they influence cellulose nanostructure. The objective of this work is to understand the influence of solvents on cellulose nanostructure with the goal of improving the degradability of cellulose nanostructure using solvents. To understand solvent interaction with cellulose, phosphoric acid was used to first solubilize cellulose (PAS cellulose) followed by adding an organic liquid or water to wash the phosphate from the system. The Flory Huggins theory was used to predict wash liquids that could favorably interact with cellulose. A favorable wash liquid was predicted to prevent the reformation of crystalline domains to yield a disrupted cellulose nanostructure, which should be more degradable. Low molecular weight alcohols and glycols were calculated to be favorable wash liquids. Washing PAS cellulose with the predicted favorable liquids yielded semi-transparent gel-like materials compared to the opaque white precipitate formed when water or unfavorable solvents were used in the wash. Fractal analysis of small angle neutron scattering (SANS) of these apparent gels indicated cellulose polymers likely have the properties of clustered rods. This partial disruption increased degradability relative to the water washed PAS cellulose. The apparent rod-like cellulose nanostructures suggested the presence of intra and interpolymer hydrogen bonding. Characterization of the hydrogen bonding network by Fourier transform infrared resonance (FTIR) indicated the gel-like material formed by ethanol washes was the result of heterogeneous interpolymer hydrogen bond cross-links. The interactions leading to gel-like materials were evaluated using Hansen solubility parameters, which predicted mixtures of ethanol and water may be most effective for disrupting cellulose nanostructure. Fractal analysis by SANS indicated 40 % ethanol/water was most effective. Similar results were obtained when 40 % ethanol was used to disrupt the cellulose nanostructure in municipal office waste (MOW). Ethanol washes increased the degradability of MOW by at least 30 % relative to conventional water washing. This is significant because increased degradability of MOW could further the development of cellulosic biofuels by reducing the amount of enzyme required to digest the material.