Biominerals often form within a matrix of biomacromolecules in high-salinity environments, yet the relationships for how macromolecules and ionic strength influence the crystallization of sparingly soluble salts (e.g., CaCO₃) are not established. Developing a physical picture of these controls is hindered by the traditional assumption that background electrolytes are inert. In this study, we investigate calcite nucleation onto two model organic matrix polysaccharides, chitosan and alginate, in a series of ionic strength solutions (65 – 600 mM NaCl). Chitosan is near-neutral (at pH 8.5) and analogous to the structural polysaccharide chitin. In contrast, alginate has a strong negative charge akin to the many anionic biopolymers in the organic matrix. By measuring the rate of calcite nucleation onto these materials and fitting classical nucleation theory to the data, we find the interfacial free energy (γ_net) and the kinetic prefactor depend upon ionic strength for both polysaccharides. The thermodynamic barrier to nucleating calcite onto alginate strongly depends on ionic strength, while calcite nucleation onto chitosan shows a similar but weaker dependence. Parallel molecular dynamics (MD) simulations were conducted to examine ion (Ca²⁺, Na⁺, HCO₃⁻, Cl⁻) and water interactions with models of a carboxylated polysaccharide and a chitosan material. The MD predictions indicate that at higher ionic strength, the polysaccharide-solution interface is increasingly stabilized by progressively higher concentrations of Na⁺ and Cl⁻. Stronger Na⁺ interactions with the polysaccharide are observed in the carboxylated system. The numbers of H₂O and HCO₃⁻ in the Ca²⁺ hydration sphere decrease with increasing ionic strength, while the number of Cl⁻ increases for both polysaccharides. The evidence suggests the increase in interface stabilization by Na⁺ and Cl⁻ increases γ_net through reductions in the polysaccharide-solution interfacial energy. We predict the effect of higher salinity is enhanced for alginate because Na⁺ interactions with COO⁻ groups make it more difficult for Ca²⁺ to displace near-surface water and/ or Na⁺. Relatively weak Na⁺-chitosan molecular interactions lead to a lesser dependence on ionic strength. Calcite nucleation rates were also measured onto chitosan in a series of sodium halide solutions (NaCl, NaBr, NaI) and onto alginate in a series of chloride salts (LiCl, NaCl, CsCl) at constant ionic strength. CaCO₃ nucleation in the presence of electrolytes with the strongest hydration properties presents the lowest γ_net. Values of γ_net increase in the order Cl⁻<Br⁻<I⁻ and Li⁺<Na⁺<Cs⁺ for nucleation onto chitosan and alginate, respectively. The findings demonstrate that background electrolytes can modulate the energy barrier to CaCO₃ nucleation through tunable effects at the polysaccharide-solution interface. / Master of Science / Skeletal structures often form within an organic matrix of polysaccharides, proteins, and lipids. An ongoing challenge is to determine the roles of these biopolymers and their surroundings during the onset and growth of biominerals. In this study, we use two characterized polysaccharide materials as models for the organic matrix where calcium carbonate (CaCO3) biomineralization takes place to investigate the role of salinity in nucleation kinetics. By measuring the rate of CaCO3 nucleation onto chitosan (neutral) and alginate (carboxylated) and fitting classical nucleation theory to the data, we find the thermodynamic and kinetic controls on nucleation rate are dependent upon sodium chloride (NaCl) concentration for both polysaccharides. Although NaCl is considered an inert electrolyte, the thermodynamic barrier to nucleating calcite onto alginate strongly depends on its concentration. Chitosan shows a similar but weaker dependence. Simulations of the polysaccharide-NaCl environment show the polysaccharide-solution interface is increasingly stabilized by Na+ and Cl-, thus increasing the difficulty for a crystal nucleus to displace surface water and ions. The effect of higher salinity is enhanced for alginate due to stronger Na+ interactions with carboxyl groups. We also find the energetics of CaCO3 nucleation onto polysaccharides in the presence of electrolytes are dependent upon type of electrolyte. The relations suggest functional groups interact with background electrolytes (e.g., NaCl) to modulate activity and function at the polysaccharide-water interface during CaCO3 crystallization processes.

From a review of the published work in the field of cycloadditions, it is evident that further research is needed to establish the mechanism of ketene cycloadditions. This work was initiated with the intent of obtaining kinetic data which will contribute to the elucidation of the mechanism of ketene cycloadditions.

The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of kinetic parameters suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-l of F6P and then donates it to protonate the leaving phosphate. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme.

O biossólido pode ser um valioso recurso ao ser utilizado em solos agrícolas; porém, um dos principais problemas de sua utilização é a presença de patógenos que podem disseminar doenças. Os principais patógenos presentes no biossólido são vírus, bactérias, protozoários e helmintos. Dentre os patógenos existentes no biossólido, os ovos de helmintos são os mais resistentes à inativação térmica e, para helmintos, os ovos de Ascaris são utilizados como indicador desses parasitas devido à comum ocorrência e resistência térmica. Dentre os processos efetivos existentes para inativar patógenos do biossólido - compostagem, secagem e tratamento térmico, digestão aeróbia termofílica, irradiação com raios beta e gama e pasteurização - este último, utilizando como fonte de calor o vapor saturado gerado a partir da queima do metano produzido em digestores anaeróbios de ETEs convencionais, é um processo de tecnologia simples, com baixo custo de implantação e operação e necessita de pequena área para implantação, sendo indicado para grandes metrópoles de países em desenvolvimento. A inativação térmica de helmintos do biossólido é o objetivo deste projeto de pesquisa. São estudadas as cinéticas de inativação térmica de ovos de Ascaris suum em água e em biossólido digerido, utilizando-se reator batelada aquecido diretamente com vapor saturado. Aplicando-se o método integral, foram determinadas a ordem das reações, as constantes específicas de morte térmica e as energias de ativação. Os ovos de Ascaris suum utilizados no trabalho foram obtidos do útero de fêmeas adultas, e o método de Yanko foi empregado para recuperação dos ovos do biossólido digerido. A inativação térmica de ovos de Ascaris em água e em biossólido digerido em processo contínuo também foi estudada por meio da modelagem matemática de um reator tubular. Os modelos propostos foram o reator tubular isotérmico com perfil de escoamento não ideal e o reator tubular com perfil axial de temperatura e escoamento tubular ideal. O primeiro foi o que melhor ajustou-se aos dados experimentais. / Biological sludge can be a valuable resource for agricultural soil conditioning. However, an important obstacle for its use is the usual presence of pathogenic organisms, capable of disease dissemination. The main occurring pathogens are virus, bacteria, protozoa and helminth. Helminth eggs are very resistant to thermal inactivation. The Ascaris lumbricoids sp. are by far the most conspicuous and resistant among helminths, reason why they have been chosen as indicator organisms for this research. The main available systems to inactivate sludge pathogens are composting, drying and thermal treatment, anaerobic thermofilic digestion, beta and gamma radiation, and pasteurization. Pasteurization through application of saturated steam, produced from burning of methane gas, generated in anaerobic digestors is a very simple technology involving low capital costs and needing relatively small areas for implementation. It can be a valuable technology to attend conditions prevailing in large metropolitan areas of industrializing countries. Thermal inactivation of helminth eggs in water and sludge is the main purpose of this investigation. Kinetics studies of thermal inactivation by saturated steam was performed using batch reactors. Application of the integral method has allowed for the determination of reaction orders, the specific constants of thermal die away as well as the activation energies. The helminth eggs (Ascaris suum) utilized have been obtained from uterus of adult females and the Yanko method was utilized for the recovery of eggs from the digested sludge. In the same way the thermal inactivation of Ascaris eggs in water and in digested sludge has been performed in continuous process by mathematical modeling of a plug flow reactor. The proposed models were the isothermic plug flow reactor with a non-ideal flow profile and with an axial temperature profile and ideal flow. The experimental data has shown a better adjustment to the isothermic plug flow reactor.

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