Chemical and Biomolecular Engineering - Theses

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    Investigating the Dynamics of Brownian Particles in Polyelectrolyte Solutions
    Ashok, Avinash ( 2021)
    The transport properties of the Brownian particles in macromolecular solutions are utilised in a wide range of industrial applications. The mobility of particles in complex solutions having characteristic length scales comparable to that of the diffusing particle tends to deviate from the Stokes-Einstein behaviour proposed for continuous medium. In recent years, particle tracking technique using high-resolution video microscopy has enabled researchers to investigate the diffusion dynamics of particles in complex fluids, particularly polymer solutions. However, the particle-macromolecule interactions and the diffusion dynamics of particles in non-adsorbing polyelectrolyte solutions near a surface or bulk remain largely unexplored research areas. Therefore, this thesis aims to identify and overcome the limitations associated with the available particle tracking techniques in order to investigate the bulk and near-surface dynamics of Brownian particles in polyelectrolyte solutions. The influence of structural interactions on the diffusion dynamics of polystyrene microparticles and gold nanoparticles in sodium polystyrene sulphonate (NaPSS) was studied using Brownian dynamic (BD) simulations, Total internal reflection microscopy (TIRM) and modified NanoSight. BD simulations and TIRM experiments were performed to explore particle dynamics near a surface, and the commercially available NanoSight was modified to investigate the bulk diffusion behaviour of particles. Through simulations, particle settlement near a surface and its confinement in structural energy wells were investigated. The simulation results helped identify the appropriate particle size, particle density and range of polyelectrolyte concentrations to overcome structural confinement and are suitable for the near-surface diffusion experiments. TIRM was combined with video microscopy to track particle motion in near-surface measurements. The spatiotemporal resolution and signal to noise ratio determine the accuracy of the measured particle trajectory. The signal to noise ratio can be improved by increasing the camera's exposure time. However, finite exposure time also creates a motion blur effect as the particle undergoes Brownian motion within the exposure time. Although researchers have explored the influence of finite exposure time in the context of bulk diffusion of particles, the exposure time effect on the position-dependent diffusion dynamics near a surface still needs to be investigated. Therefore, BD simulations and TIRM experiments were combined to address the influence of exposure time on estimating the hindered diffusivity of a microparticle undergoing Brownian motion near a surface. The result showed that the ratio of exposure time to frame rate affects the measured near-surface diffusion dynamics. The concept of motion blur coefficient was implemented into the diffusion calculation to match the experimental results with the theoretical model. The bulk diffusion dynamics were studied by customising the commercially available particle size characterisation instrument NanoSight as most of the available techniques do not directly track the Brownian motion of particles and rely on the scattering signals or are limited to tracking fluorescently labelled particles. The motion of micro and nanoparticles in bulk was tracked successfully using the modified instrument. Using the mean squared displacement data, two modes of diffusion dynamics were observed in the NaPSS solution. At a small time scale, particles followed sub-diffusion, and at a long time scale, Fickian diffusion was observed. The range and magnitude of the diffusion modes were dependent on NaPSS concentration and particle size. Detailed analysis of the time scale range and particle diffusivity also showed that particle dynamics in polyelectrolyte solution varied significantly from those reported in the literature using a non-ionic polymer. The observed bulk dynamics were compared to the confinement effects and particle macromolecule interactions near surfaces by measuring surface interactions and the near-surface of micro and nanoparticles in NaPSS solution using TIRM. While the surface interaction of microparticles followed the semi-empirical structural force model, for nanoparticles with a size similar to that of NaPSS chains, significant distortion in the oscillatory energy profile was observed. The absence of well defined structural energy wells for nanoparticles until higher concentrations of NaPSS are reached, compared to the case of microparticles, indicated that the ordering of polyelectrolyte chains might not be as effective. This may be attributed to either the scaling of the structural force with particle radius or the similarity in the length of the particle radius and the size of the depletant. The hindered diffusion profile of microparticles was explored, and the profiles were highly influenced by the structural force, particularly at small separation distances. Simulation results showed that investigating the range of diffusive time and length scales of Brownian particles near-surface requires a large amount of data at very high spatiotemporal resolutions. Therefore, the near surface diffusive behaviour was qualitatively compared with the bulk diffusion behaviour by performing evanescent wave dynamic light scattering analysis on the intensity autocorrelation functions. The results confirmed two relaxation time scales on particle diffusion even with noise challenges due to sampling at long times. The intensity autocorrelation data was further examined at a short time scale, and an average hindered diffusion coefficient was extracted, which also showed sub-diffusive characteristics as observed in bulk.
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    Unimolecular Reactions of Resonantly Stabilized Radicals in Combustion
    Adewale, Rasheed Adedamola ( 2021)
    Despite years of concerted effort, the chemical mechanisms and kinetics detailing PAH formation are yet to be fully characterized. Resonantly Stabilized Radicals (RSRs) are of special consideration among PAH progenitors in flame chemistry due to their relative stability which leads to their build-up to comparatively higher concentrations and their ability to regenerate in hydrocarbon flames makes them available for molecular growth reactions. This thesis has applied fundamental chemical theories viz ab initio calculations and master equation simulations to gain more understanding of selected RSR reactions. Recent experimental data reveals that the resonantly stabilized c-C5H5 and l-C5H5 radicals can fragment to C5H4 + H aside from the traditionally reported C3H3 + C2H2 dissociation pathways. This makes it important to include these new dissociation channels in kinetic models for better flame predictions and to keep the reactions of c-C5H5 and l-C5H5 RSRs up to date with experimental findings, in kinetic models. As such, Chapter 4 of this thesis investigated the C5H5 potential energy surface and performed RRKM/Master Equation simulations to obtain kinetic data for the C5H4 + H reactions. It was observed that the C3H3 + C2H2 is the dominant fragmentation pathway with the C5H4 + H becoming important at temperatures between 900 – 2000 K. The developed rate parameters, coupled with recent literature kinetic data for relevant reactions, were used to update a kinetic model for toluene flame. The predicted results, especially for the resonantly stabilized cyclopentadienyl (c-C5H5) radical, were in good agreement with experimental data obtained from literature, for a low-pressure toluene premixed flame. The developed and presented detailed kinetic model will aid the development and inclusion of 1-vinylpropargyl (l-C5H5) and other C5Hx reactions in combustion studies. Chapter 5 of this thesis proposes a pathway to the formation of a recently detected C7H7 isomer, 3-ethynylcyclopentenyl (3ecpr), through the l-C5H5 + C2H2 reaction, using quantum chemistry techniques and RRKM master equation modeling. The calculated ionization energy for the formation of the first triplet state is in the range of 9.1 – 9.3 eV, which matches the appearance energy of a mystery C7H7 isomer seen in VUV photoionization experiments on the C3H3 + C2H2 reaction cascade. The low appearance energy for the ground state singlet cation (6.9 eV), and sharp photoionization onset apparent in Franck-Condon simulations, explain the absence of this state in these experiments. The C3H3 + C2H2 reaction produces the l-C5H5 isomer in experiments at 800 K and 8 Torr, and the master equation kinetic modeling done in this thesis illustrates that under these conditions the further addition of acetylene to l-C5H5 produces stabilized 3ecpr as a dominant reaction product. Simulations carried out at combustion relevant temperatures and pressures demonstrate that the l-C5H5 + C2H2 reaction will produce both 3ecpr and ethynylcyclopentadiene + H. The present work now allows for further development of l-C5H5 and C7H7 chemistries and their adequate characterization in detailed chemical kinetic models. The oxidation and molecular growth reactions of a larger resonantly stabilized radical (RSR), alpha-styryl (C6H5CCH2), were studied in Chapters 6 and 7 respectively. Regarding the addition of O2 to C6H5CCH2 reaction, it was predicted to proceed to multiple product channels (e.g., benzoyl + CH2CO, phenacyl radical + O, benzyl + CO2, benzoxyl radical + CO, benzaldehyde + HCO, isobenzofuranone + H) and it is pressure-dependent at temperatures below around 1300 K. This is contrary to what is obtainable in current kinetic models where the reaction is represented by an estimated high-pressure rate coefficient with a single product channel, benzoyl + CH2CO. Although the reaction population is dominated by benzoyl + CH2CO, phenacyl radical + O is predicted to be the main product at T > ~2650 K. The decomposition reactions of selected products of the C6H5CCH2 + O2 was also studied to provide kinetic data for their reactions in combustion studies. Phenacyl was found to predominantly dissociate to benzyl + CO while isobenzofuranone and benzofuranone have benzaldehyde and quinone methide has main decomposition products respectively. The newly calculated rate coefficients were tested in a styrene flame and were found to improve the concentration predictions of benzyl, phenol, and ortho-benzyne. Similar to the C6H5CCH2 + O2 reaction, the C6H5CCH2 + C2H2 is described with ‘simple’ high-pressure limit estimates in combustion models with naphthalene + H as the only products. The kinetic simulations performed in this work predict that at least two other product channels – methyleneindene + H and azulene + H are feasible. Furthermore, methyleneindene + H is predicted to be the dominant product and not naphthalene + H. Following the testing of the obtained rate data in a rich styrene flame, it was identified that reactions of methyleneindene are currently scarce in kinetic models, thereby inhibiting the adequate interplay of methyleneindene with other species in kinetic models. This makes it important to investigate other reactions of methyleneindene e.g., interconversion with other C10H8 species, molecular growth pathways, and oxidation reactions.
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    Atmospheric Degradation of Heteroatom-Containing Volatile Organic Compounds
    Ren, Zhonghua ( 2021)
    In this thesis, ab initio modelling and RRKM theory/master equation kinetic simulation are used to study the atmospheric degradation of several important heteroatom-containing volatile organic compounds (VOCs), piperazine (Pz), tetramethylsilane (TMS), hexamethyldisiloxane (HMDSO) and trimethylsilyl formate. Firstly, atmospheric oxidation of the nitrogen-containing VOC piperazine, initiated by OH, has been studied. The Pz + OH reaction is found to proceed at around the capture rate, consistent with experimental data, with abstraction predominantly from C—H sites, forming an alkyl radical (PzC). The subsequent reaction kinetics of carbon-centred Pz radicals with O2 are also studied, so as to determine the first-generation oxidation products. We find that the PzC radical predominantly reacts with O2 to produce a cyclic imine product + HO2 under tropospheric conditions, with the stabilized peroxyl radical formed as a minor product. Subsequent reaction of the peroxyl radical with NO produces an alkoxyl radical that can react with O2 to yield a cyclic amide or undergo unimolecular ring opening followed by a second O2 addition / HO2 elimination step to produce CH2=NCH2CH2NHCHO. Next, the atmospheric chemistry of tetramethylsilane has been investigated. Under tropospheric conditions the radical (CH3)3SiCH2 reacts with O2 to produce a stabilized peroxyl radical which is expected to ultimately yield the alkoxyl radical (CH3)3SiCH2O. At combustion-relevant temperatures, however, a well-skipping reaction to (CH3)3SiO + HCHO dominates. Importantly, the (CH3)3SiCH2O radical is predicted to rearrange to (CH3)3SiOCH2 with a very low reaction barrier, enabling an auto-oxidation process involving the addition of a second O2. Subsequent oxidation reaction mechanisms of (CH3)3SiOCH2 have been developed, with the major product predicted to be the trimethylsilyl formate (CH3)3SiOCHO, an experimentally observed TMS oxidation product. The production of substantially oxygenated compounds following a single radical initiation reaction has implications for the ability of VOSiCs to contribute to ozone and particle formation in both outdoor and indoor environments. Besides, we have studied the photooxidation of hexamethyldisiloxane. Reaction of alkyl radical (CH3)3SiOSi(CH3)2CH2 with O2 is found to predominantly produce peroxyl radical (CH3)3SiOSi(CH3)2CH2O2, followed by RO2 + NO chemistry to yield alkoxyl radical (CH3)3SiOSi(CH3)2CH2O. Then the alkoxyl radical can rearrange to alkyl radical (CH3)3SiOSi(CH3)2OCH2 via a similar mechanism seen in TMS chemistry. Oxidation of (CH3)3SiOSi(CH3)2OCH2 will ultimately produce (CH3)3SiOSi(CH3)2CH2O. Subsequently, three possible reactions that yield the experimentally observed product (CH3)3SiOSi(CH3)2OCHO have been discussed with (CH3)3SiOSi(CH3)2OCH2O + O2 reaction predicted as the dominant. Finally, we have studied the atmospheric degradation of trimethylsilyl formate. Silyl formates are major products in the atmospheric oxidation of volatile methylsiloxanes (VMSs) and CO2 fixation by hydrosilylation. Yet, the atmospheric chemistry of silyl formates remains less studied. Here, HC(=O)OSi(CH3)3 is selected as a model and its atmospheric oxidation initiated by OH has been investigated by both quantum chemistry calculations and master equation simulations. The reaction predominantly proceeds by H abstraction from the aldehyde group to yield C(=O)OSi(CH3)3 which can then associate with O2 to form peroxyl radical O2C(=O)OSi(CH3)3. According to kinetic calculations, the peroxyl radical stabilization is predicted to be important. Subsequent reactions of alkoxyl radical OC(=O)OSi(CH3)3 have been investigated. Instead of decomposition to small fragments, it will lead to highly oxygenated and less volatile products, NOxOC(=O)OSi(CH3)3 and HOC(=O)OSi(CH3)2CHO, which may contribute to the secondary organic aerosols and cause air pollution problems.
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    The role of anti-solvents, nanoparticles and nanocatalysts in energy-efficient CO2 capture
    Sheikh Alivand, Masood ( 2021)
    Rising CO2 emissions from global industrial sources have strengthened international concerns and scientific endeavors to reduce the impact of anthropogenic climate change. The Paris Agreement in 2015 focused attention on reducing the growth in CO2 emissions over the coming decades through a variety of mechanisms. One viable approach is carbon capture and storage (CCS), which is strongly reliant on feasible CO2 capture technologies, as this will enable international governments to meet their Paris Agreement targets, especially from the industrial sector. However, most of the CO2 capture technologies, particularly chemical CO2 absorption as the most viable approach, are energy-intensive and suffer from a highly expensive cost of operation. Therefore, the current developed technologies for CO2 capture have not been extensively deployed as a large-scale technology. Different approaches, including non-aqueous phase change solvents, CO2 capture using nanofluids, and employing heterogeneous catalysts have been reported in the literature to overcome the high energy demand of CO2 absorption-desorption processes. These suggested technologies have not been shown to be efficient, and therefore not economically acceptable for industry. Since most of these are in the initial stages of their development, it was recommended by literature that these areas should be further assessed which is the scope of the present thesis. The aim of my research was to present new and facile techniques to decrease the energy requirement of the current CO2 absorption-desorption processes. In the first approach, H2O/DMF mixture was introduced as a binary solvent to prepare potassium glycinate solutions (i.e., GlyK) for CO2 absorption. The presence of DMF enabled the solution to exhibit phase-change behavior with three different phases. The effect of the DMF:H2O ratio on the volume of different phases was also investigated. The investigation showed that the upper liquid phase was completely free from CO2-containing species like carbamate and bicarbonate which was a unique achievement. Hence, the upper liquid phase can be recycled to the absorption column whilst the rest of the solvent is regenerated, resulting in a substantial energy saving throughout the cyclic CO2 absorption-desorption operation. The results displayed that the GlyK-60 solvent with DMF:H2O volume ratio of 60:40 had a very high CO2-free phase volume (63%) and 59.1% reduction in relative heat duty compared to the conventional aqueous GlyK solvent. In the second results chapter, a new methodology was presented to tailor the physicochemical properties of metal-organic frameworks (MOFs) for the synthesis of water-dispersible core-shell nanocatalysts with ease of use. The characterization exhibited that functionalized nanoclusters (Fe3O4-COOH) effectively induce missing-linker deficiencies and fabricate mesoporosity during the self-assembly of MOFs, providing high proton donation capability and unique catalytic properties. The nanomaterials obtained drastically reduced the energy consumption of CO2 capture by 44.7% using only 0.1 wt.% nanocatalyst, which is a 10-fold improvement in efficiency compared to heterogeneous catalysts. Lastly, I reported a facile and eco-friendly approach for the synthesis of water-dispersible Fe3O4 nanomaterials coated with a wide range of amino acids (12 representative molecules) in aqueous media. It was demonstrated that the functional groups and acidic properties of the prepared materials can be easily tuned, making these nanomaterials efficient nanocatalysts in energy-efficient CO2 capture plants with ease-of-use, at very low concentrations (0.1 wt.%) and with extra-high efficiencies (up to 75% energy reductions). The performance of acidic Fe3O4 nanomaterial was also tested in a range of solutions, including amino acids (i.e., short and long-chain) and amines (i.e., primary, tertiary, and primary-tertiary mixture) which resulted in 46.3-67.4% relative heat duty for amino acids and 24.6-57.6% for amine solutions. Due to the potential, simplicity and superiority of the presented techniques in this thesis, particularly their compatibility with renewable energy resources for cyclic operation, these technologies are expected to provide a new pathway for the development of energy-efficient CO2 capture technologies and allow for a more affordable CO2 capture process.
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    Application of Advanced Fluorescence Imaging Techniques for Intracellular Tracking of Nano-biomaterials
    Radziwon, Agata ( 2021)
    The engineering and intracellular delivery of nanoparticles with tailored structural, functional and therapeutic properties is challenging due to the interactions of nanomaterials with complex and dynamic biological systems. Additionally, the clinical translation of nanoparticle-based chemotherapeutics is hampered by the poor capacity of 2D cell monolayer culture to mimic in vivo tumour microenvironment and cell-cell interactions. To overcome these biological barriers and enable the clinical translation of nanoparticles, a thorough investigation of nanoparticle-cell interactions in complex biological environments is of paramount importance. For this purpose, novel advanced fluorescence techniques enable the study of nanoparticles structure and functional properties inside the cells with improved spatial and temporal resolution. Additionally, the development of complex 3D cell culture systems mimicking tumour tissue could provide a novel method to predict the in vivo behaviour of nanoparticle-based chemotherapeutics and cellular response to the treatment. Herein DNA- and sugar-based nanoparticles have been developed as platforms for the detection of molecular targets and delivery of drugs within cells and in complex biological settings. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localization microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the nanocomplexation of glycogen with albumin. The intracellular trafficking and the activity of drug-loaded nanoparticles were investigated in 2D and 3D static and dynamic cell culture systems. The biological activity of glycogen-albumin nanoparticles was investigated in a 3D tumour microtissue obtained by co-culturing BT474, NIH-3T3 and RAW264.7 cells in a U-Cup perfusion bioreactor device. The interactions of glycogen-albumin nanoparticles with peripheral blood mononuclear cells isolated from human blood and nanoparticles in vivo biodistribution in mice were also analyzed. This study aims to gain an understanding of the bio-nano interactions in various biological systems and highlights the importance of combining multiple fluorescence techniques and complex models for monitoring the intracellular behaviour of nanomaterials and accurately predicting their in vivo behaviour.
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    Enzymatic Synthesis of Lactose-Based Bioactive Molecules
    Karimi Alavijeh, Masih ( 2021)
    Functional food products and nutraceuticals have attracted considerable attention as they provide potential health benefits in humans. Global bioactive ingredient market data shows that functional food products have turned into a major business, driving a need for efficient and sustainable routes to produce defined compounds with specific health promoting properties. Bioactive lactose-based molecules, including human milk oligosaccharides (HMOs) and galactooligosaccharides (GOS), are examples of functional ingredients that can be supplements to food products. N-Acetyllactosamine (LacNAc) is a structural component of many biologically active compounds such as HMOs, glycoproteins, sialylated carbohydrates and poly-N-acetyllactosamine-type oligosaccharides. Currently, despite excellent research performed to study LacNAc-based building blocks, their commercial production is in an early stage. Commercially available starting substrates can facilitate industrial-scale biochemical production of these value-added ingredients. As an abundant and inexpensive substrate obtained from whey, lactose is an ideal starting substrate that can be used in both in vivo and in vitro biochemical syntheses. The aim of this thesis is to synthesize LacNAc-related molecules from whey-derived substrates, including lactose and casein glycomacropeptide (CGMP), using glycoside hydrolases. As a starting point, a comprehensive analysis of the importance of LacNAc and existing production strategies of both LacNAc and important LacNAc-based structures, including sialylated LacNAcs, as well as poly- and oligo-LacNAcs was compiled. A large-scale process was then designed for the enzymatic synthesis of LacNAc from lactose and N-acetylglucosamine (GlcNAc) based on the use of thermostable B-galactosidases from Bacillus circulans (BgaD-D), Thermus thermophilus HB27 (TtB-gly) or Pyrococcus furiosus (CelB). Downstream purification of LacNAc was simulated based on anion-exchange chromatography, an activated charcoal-Celite column, GlcNAc crystallization and an activated charcoal-Celite column, as well as selective crystallization. The effect of enzymatic yield, lactose concentration and acceptor to donor ratio on the project costs was discussed. The results showed that the process based on BgaD-D gave the best economics among the enzymes examined. In addition, the minimum LacNAc sales price can be reduced to $2 per gram by the use of selective crystallization as the most economically viable purification step. For most processes, GlcNAc mainly contributed to the raw material costs, while methanol contributed 72% of these costs for the process based on an activated charcoal column. The methanol consumption can, however, be reduced by 73% using a crystallizer for GlcNAc separation before the chromatography column. In the second part of this thesis, the transgalactosylation kinetics of the B-galactosidase from Bacillus circulans in the presence of cations present in dairy whey systems, namely calcium, magnesium, sodium and potassium, was investigated using both molecular modeling and quantitative experimental methods. This study indicated that hydrolysis and transgalactosylation reaction kinetics were not significantly affected at low concentrations of divalent cations (Ca2+ or Mg2+) or up to 100 mM of monovalent cations (Na+ or K+) compared to a control reaction. In contrast, high concentrations of calcium and magnesium (100 mM) triggered enzyme aggregation and progressive formation of an insoluble protein network resulting in the loss of enzyme activity. This decrease in the enzyme activity with time led to significant changes in the enzymatic yield and selectivity. The calculated electrostatic surface potential map of the enzyme was indicative of dominant negatively charged areas, which can interact with calcium and magnesium as strong salt-bridge forming cations. The docking position of these divalent cations were also predicted. This study presents a potential way to regulate the Bacillus circulans B-galactosidase reaction pathways by addition of divalent cations through the formation of protein aggregates. The next part of this thesis deals with the use of layer-by-layer deposition as a versatile technique for immobilizing the B-galactosidase in aqueous solutions under mild conditions. Commercially available silica particles were used as the base support in conjunction with robust pairs of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) to encapsulate the B-galactosidase into the layers. The effect of multilayer films on the immobilized B-galactosidase stability and catalytic activity was studied in detail. Hydrolytic activity measurements indicated a significant decrease in the enzyme activity after immobilization. In addition, the higher the enzyme dosage applied during the immobilization process, the greater the activity reduction observed. Molecular analysis was further performed to study the possible interactions (electrostatic, covalent and protein-protein interactions) during this encapsulation method, that can contribute to the enzyme hydrolytic activity reduction. In contrast, the immobilized B-galactosidase was able to produce more LacNAc compared to the free counterpart. Moreover, the thermal and operational stability of the enzyme was substantially enhanced after the immobilization, allowing the successful recovery and reuse of the enzyme in consecutive cycles. The analysis of a large-scale process based on the immobilized B-galactosidase demonstrated significantly improved sustainability and economics. In the final part of this thesis, LacNAc directly produced and purified in cheese whey (as the acceptor) and whey-derived casein glycomacropeptide (CGMP) (as the donor) were used to synthesize 3’-sialyl-N-acetyllactosamine (3’-SLN) using a sialidase from the nonpathogenic Trypanosoma rangeli mutated with 15 amino acid substitutions (Tr15). The time-course study of the reaction was performed under different conditions in terms of acceptor to donor ratio, CGMP concentration and enzyme concentration. A high yield of 3’-SLN (75%) based on the available bound alpha-2-3-sialic acid in CGMP was obtained. This demonstrates the potential of this method for industrial applications as compared to sialyltransferase-catalyzed reactions requiring expensive nucleotide sugars. Furthermore, the reaction was successfully modeled using either a mechanistic kinetic analysis or a machine-learning-based approach, using an optimized artificial neural network. This reaction study showed the high trans-sialylation activity of Tr15 in the reaction with CGMP and LacNAc as sialyl donor and acceptor, respectively. In addition, as this enzyme is obtained from nonpathogenic species, it would be of more interest for pharmaceutical and food applications, compared to other sialidases that are virulence factors for pathogenic species.
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    Engineering Nanocapsules with Tunable Physiochemical Properties for Controlled Bio−Nano Interactions
    Li, Shiyao ( 2021)
    The rapid development of nanoparticle engineering has extended into a wide range of bio-medical applications. Metal-phenolic network (MPN) capsules, which are formed upon coordination of polyphenols with metal ions, have emerged as a promising class of particle-based drug delivery systems, due to their tunable physiochemical properties and drug-loading capability. For example, the use of poly(ethylene glycol) (PEG)-conjugated polyphenol to coordinate with metal ions affords PEG-MPN capsules that show reduced nonspecific cellular associations in vitro when compared with MPN capsules prepared from tannic acid (TA) as a polyphenol. However, to date, the literature studies on MPN capsules have generally focused on the preparation of MPN capsules on the micrometer scale, which is not within the optimal size range of nanoparticle-based drug delivery systems. It has been a challenge to engineer MPN capsules on the nanometer scale with controllable size. Therefore, engineering MPN nanocapsules with controllable physiochemical properties and subsequently studying the interactions between the nanocapsules and different biological systems are important towards effectively advancing MPN capsules in biomedical applications. In the present thesis, MPN nanocapsules with sizes between 50 to 150 nm were engineered through template-assisted self-assembly. The effects of capsule size on the bio-nano interactions between PEG-MPN nanocapsules and different biological systems were investigated through in vitro cell association assays, ex vivo blood assays, and in vivo circulation and biodistribution studies. The PEG-MPN nanocapsules of 50 nm showed reduced association with leukocytes (up to 70%) and longer circulation time in vivo (4 h vs. 1 h) than the 150 nm PEG-MPN nanocapsules. The PEG-MPN nanocapsules were then endowed with targeting capability to target tumor cells by functionalizing the nanocapsules with anti-PEG-anti-epidermal growth factor receptor (EGFR) bispecific antibodies. The obtained PEG-MPN-EGFR showed specific association with EGFR-overexpressed MDA-MB-468 human breast cancer cells in vitro. Although the targeting ability of PEG-MPN-EGFR was largely limited in the presence of human blood (particle association reduced to 1/9 of the in vitro results; that is from ~81 to ~9 nanocapsules per cell), it was restored in washed blood conditions (where the human plasma was removed from the blood). These results were comparable to that of the in vitro experiments, demonstrating the important role of human plasma in regulating the bio-nano interactions. Finally, the effect of different protein corona on immune cell-particle interactions was investigated. TA-based MPN nanocapsules were precoated with a protein corona composed of different serum proteins (bovine serum albumin, fetal bovine serum, and bovine serum), and the association between the precoated MPN nanocapsules with leukocytes in human blood was analyzed using mass cytometry. Precoating the nanocapsules with serum proteins effectively reduced association with leukocytes, in which precoating the nanocapsules with fetal bovine serum reduced the capsule association with neutrophils and monocytes up to 90% compared with nanocapsules without precoating. Proteomics analysis of the protein corona composition suggests that the enrichment of immunoglobulins is positively correlated with the increased leukocyte association of the nanocapsules. Overall, MPN nanocapsules with controlled physiochemical properties were engineered and the interactions between the nanocapsules with different biological systems were studied.
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    Enhancement of CO2 capture and utilization by microalgae
    Xu, Xiaoyin ( 2021)
    CO2 capture and utilization has been a hot topic for decades. Photoautotrophic microalgae production is a carbon-friendly industry that converts CO2 to biomass with value-added proteins and bio-oils. This study aims at facilitating the microalgal fixation of CO2 via two different methods. Firstly, the thesis investigates a liquid-liquid membrane contactor to deliver CO2 captured from large point sources by a chemical solvent to the microalgae culture. The CO2 mass transfer coefficients of two membrane modules with varied membrane areas were studied. The solvent flow was controlled by pH feedback. The microalgae productivity and CO2 utilization efficiency with different membrane modules and CO2 supply conditions were compared. Three methods to immobilize carbonic anhydrase (CA), as a CO2 capturing enzyme are then investigated. These methods are layer-by-layer electrostatic adsorption, hollow fiber entrapment, and tannic acid-mediated assembly. The methods are compared based on enzyme activity, cyclic stability, and their enhancement to microalgae production. A fourth immobilization method combining CA-glutaraldehyde cross-linking and buoyant calcium alginate beads encapsulation is developed and used to demonstrate the use of interfacial CA catalyzed atmospheric CO2 fixation by microalgae. The CA beads were characterized with microscopic, protein bonding and activity assays. The biomass productivity, dissolved carbon concentration stability, culture water loss and enzyme cyclic performance were monitored, and the cost was calculated to assess the potential of applying immobilized CA in the microalgae industry. It was found that CA-encapsulated buoyant alginate beads provided a recyclable, stable and effective additive to the microalgal pond. Finally, a study is conducted on inhibitive effects of the microalgae medium on CA activity by the Wilbur-Anderson assay. A substitute culture medium was suggested based on the assay results and the performance of CA in the new medium was studied.
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    The behaviour of almond proteins in purified, minimally processed and complex food systems
    Devnani, Bhanu ( 2021)
    A growth in consumer preferences for plant-based diets has triggered the utilisation of plant ingredients in a variety of food and beverage applications, including substitutes to dairy products. The majority of commercial plant-based dairy alternative products, however, have reduced protein content compared to dairy products. Such protein plays an important role in dairy products, including yoghurt and cheese, where it imparts functionality, such as gelation and texture. These differences in protein content and properties in plant-based systems, compared to dairy products, may be responsible for the commonly encountered technological challenges, such as the replication of the texture and mouthfeel. With an attempt to fill these gaps, this thesis aimed to explore the potential of almond as an ingredient to develop novel high protein products. First, commercial almond-based yoghurt alternatives were studied to understand the role of almond proteins in these complex food systems and establish the difference between almond and dairy systems. Next, almond proteins were derived in the form of minimally processed extracts or purified isolates to develop almond milk and almond protein isolate. The effect of processing parameters, including temperature and pH, on the fundamental and functional properties of these protein extracts was then studied, with the objective of gaining deeper mechanistic insights into almond protein gelation. First, a range of objective instrumental techniques were used to assess whether commercial almond-based yoghurt alternatives behaved differently to soy and dairy yoghurts. All almond-based yoghurt alternatives contained added stabilisers and were lower in protein compared to dairy yoghurt (less than 2.7 wt percent in almond vs 5 wt percent in dairy). The interconnected protein network, which is known to structure dairy yoghurt, was absent in almond based yoghurts. Instead, these systems appeared to be flocculated and contained swollen starch granules, protein and fat particles/ aggregates. As a result, the almond yoghurts had lower colloidal and structural stability, in comparison to dairy yoghurt and also differed in their rheological and tribological properties. This study highlights the areas that require attention to further optimise almond yoghurts if product developers wish to mimic the properties of dairy yoghurt. Second, the effect of thermal treatment (45–95 degree Celsius for 30 min) on the structure of almond proteins extracted in minimally processed almond milk was assessed, as the unfolding and association of these proteins in response to heat, and its impact on colloidal stability and gelation of almond milk was not well understood. This temperature range was chosen based on the wide range of denaturation temperatures reported previously for almond proteins (45–115 degree Celsius). Above 55 degree Celsius, protein surface hydrophobicity and particle size increased, while alpha helical structure decreased, reducing the stability of skim or full fat almond milk. Fractal protein clusters were observed at 65–75 degree Celsius and weakly flocculated gels with a continuous protein network occurred at 85–95 degree Celsius, resulting in gels with high water holding capacity (approximately 70 percent) and a strength similar to dairy gels at similar protein concentrations of approximately 4 wt percent. The presence of almond fat increased the gel strength measured but led to a more heterogenous microstructure. The elasticity of almond gels could also be increased approximately 25 times with a threefold increase in protein concentration (i.e. from 3.6 percent in skim almond milk to 10.8 percent post concentration). This study provided a better understanding of the heat sensitivity of almond milk proteins and revealed temperatures that are critical to almond milk processing for a variety of applications, including heat treatment to induce denaturation prior to almond yoghurt formation or heat-induced gelation to form products like almond tofu/cheese. Third, the behaviour of purified almond protein isolate, containing predominantly amandin protein, was examined under conditions of neutral and acidic pH (pH 7 and 4). The isolate was highly soluble (70-80 percent) at either pH. An increase in acidity led to protein unfolding, an increase in random coil structure and the appearance of lower molecular weight proteins, potentially due to acidic and/or proteolytic hydrolysis. These structural changes at pH 4 increased the capacity for foam formation and foam stability, increased viscosity and led to concentration and age dependent thickening. Gels, similar in strength but with distinct microstructures and properties, were obtained following heating. At pH 7, a particulate type gel with an interconnected protein network was formed, while the gel produced at pH 4 had a dense continuous protein matrix. The gels differed in their susceptibility to chemical disruption, suggesting different underlying molecular interactions. This study illustrates the potential utility of almond proteins in foaming, thickening and gel formation and how almond preparations can be tuned by varying pH and temperature to obtain a range of tailored products with desired properties. Overall, these studies have increased our understanding of the response of almond proteins to process variables that are essential for both product and process development, performance and stability. Interconnected protein-based gel networks could be developed using either minimally processed or purified almond protein systems where the protein concentrations were equal to or greater than 4 wt percent. The formation of such gels and their pH tuneable properties may assist the formulation of novel almond based gelled vegan products, that are at par with dairy both in terms of protein content, functional performance and consumer acceptability.
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    Protocol development for chemical removal in water treatment and water recycle
    Pang, Hongjiao ( 2021)
    Water security and scarcity, driven by a growing population, increased urbanization and a changing climate, are becoming critical issues worldwide. Augmentation of water supply with recycled wastewater as an alternative water source to meet the increasing clean water demand is becoming more common, with significant benefits as a rainfall and climate independent water source. The myriad of pathogens and emerging chemical contaminants in secondary treated wastewater means that additional treatment using a multi-barrier water treatment system is needed to achieve potable quality recycled water. However, a dominant issue to the barrier selection is an inability to predict the performance for the removal of contaminants. A key hindrance to barrier operations at an industrial scale is the cost of ongoing compliance testing of contaminants and performance validation to ensure that the contaminants in feedwater are removed to below guideline levels. In the case of pathogen removal, use of a Critical Control Point (CCP) approach for operations, along with validated barrier credits, allows predictive performance of a barrier (i.e. UV, Ozone, Reverse Osmosis and Chlorine). This approach has been developed for each class of pathogens in water treatment and water recycling (water recycling is a term used herein to describe the process whereby wastewater is treated for a range of beneficial uses, including surface or groundwater supplies in landscape or agricultural irrigation, industrial applications, and potable reuse) and has proven highly successful globally. The CCP protocol developed for each barrier significantly reduces the need for regular (daily) compliance testing using complicated analysis since barrier integrity and compliance validation are inextricably linked. However, for chemical removal, there is still not a risk-based approach where a barrier is operated with validated credits for chemical removal. Analogous to the pathogen risk reduction approach, there are an increasing number and diversity of emerging chemical contaminants and an appropriate approach that allows chemicals to use a similar barrier credit and CCP criterion as pathogens is envisaged. The starting point is an intimate knowledge of the relationship between the characteristics of a chemical and the key removal mechanism of a particular barrier. Therefore, this thesis focuses on extending the CCP approach along with mechanistically relevant barrier credits to chemical contaminants. Development of surrogates of barrier performance inclusive of on-line monitoring and a decision tree of the type of chemical and the associated removal credits for each barrier, linked to the CCP approach, that can be easily used in the field, was also of interest. In the existing literature, Ozone and Reverse Osmosis (RO) have been mentioned as potential effective barriers for removing a wide spectrum of chemical contaminants in water treatment and water recycling systems. Thus, Ozone and RO were selected as targeted barriers in this thesis to develop the chemical credit protocol in the first instance. Development of surrogates of barrier integrity relevant to the chemical removal then provides the basis of a CCP approach. To identify the barrier credits for as many chemical contaminants as possible, so as to develop a generalized chemical credit system, the removal performance of 992 trace organic chemicals was quantitated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analysis was applied across either an Ozone or RO barrier to validate the barrier performance to each class of chemicals.A log reduction value (LRV) was used to represent the removal efficiency and chemical classifications assigned that were based on the mechanisms of removal of the barrier. In this thesis, the chemical credit protocol is developed for the removal of emerging chemical contaminants through an Ozone and RO barrier. The protocol was validated in full-scale operations and a surrogate of barrier performance was investigated as a precursor to the development of a fully integrated CCP framework. Furthermore, a novel and sensitive surrogate method for the determination of per-fluorinated carboxylic acids (PFCA) in solution has been developed with the potential for utilization in simple laboratories and field laboratory scenarios. These molecules have a low guideline concentration for exposure and environmental discharge in many countries. For the Ozone barrier, removal credits for a range of chemical types (linked to the removal mechanisms) were established using bench-scale operations. The work explored a wide range of operational conditions. It allowed a number of proposed chemical classifications to ozone response to be tested. A conservative predictive framework for the removal of each class of chemicals was then proposed. The framework included both an ozone residual concentration by time (CT value, mg min/L) and O3/TOC (total organic carbon) mass ratio relative to the instantaneous ozone demand (IOD) with the development of chemical classifications according to mechanisms of removal as defined through a susceptibility to ozone and a specific chemical structure. This classification is considered generic to all organic molecules. Measurement of UV254 and TOC were also tested as potential online surrogates of ozone barrier performance, providing the basis for the development of a fully integrated CCP framework. Full-scale validation of the approach was undertaken at the Eastern Treatment Plant, Melbourne, Australia. A CT value and O3/TOC ratio were, in combination, shown to be necessary for the prediction of the removal efficiency of a given chemical through the ozonation process. In the case of pathogens, either of these two parameters is often taken to be adequate. For the RO barrier, a barrier credit framework was developed for a polyamide RO membrane by focusing on the removal of a wide spectrum of chemical contaminants along with mechanistically relevant barrier credits to RO under specific but industry-relevant operational conditions. The site used for performance validation was a plant producing high-quality water for industrial and agricultural reuse. A comprehensive chemical classification inclusive of molar volume, molecular charge and molecular water-oil partition characteristics was utilised in the barrier credit framework. With such a system, both the LRVs for individual chemical contaminants through routine testing and the conservative LRVs that can be consistently verified for the class of chemicals were determined. Conductivity was validated as an efficient surrogate for chemical removal for specified operational parameters such as permeate flux and recovery rate. Meanwhile, the concept of using bioassay toxicology data was introduced as a comparison with chemical detection data. It was confirmed as an appropriate first screen or hurdle test to indicate treated water quality and barrier performance. This is consistent with trials being conducted in California, USA, and provides the potential for cheap on-site hurdle testing that is highly complementary to off-site chemical analytics. Additionally, a new analytical test for per-fluorinated carboxylic acids (PFCA), an emerging group of chemicals of concern, was developed. Analysis of these chemicals commonly requires professional operational skills and expensive instrumentation. Such an approach is not amenable to on-site testing. The new methodology shows a high sensitivity of detection of PFCA with the potential for utilization in simple laboratories and field laboratory scenarios. The method is based on solid-phase extraction followed by simple heat-based derivatization and analysis with a HPLC-UV system or just a UV-Vis spectrometer to measure each PFCA compound or the combined/total PFCA compounds in water. Sub-ppm determination of PFCAs was achieved for analysis using both HPLC-UV and UV-Vis spectrometry. The sensitivity is close to the guideline limits for water in many contaminated soil operations. Overall, extending the barrier credits framework from pathogens to chemicals is an obvious step with potential benefits for all water treatment facilities world-wide. This thesis confirms the barrier credit system as an effective approach to assist in improving water quality management through the utilisation of chemically contaminated water as a feed source. To our knowledge, the use of the chemical credit framework in water treatment and water recycling is predominately confined to pathogen mitigation. This work provides a framework for the adoption of CCP criteria integrated with barrier credits for the operational removal of chemical contaminants. This will facilitate system design and help operators understand the expected performance of each barrier since the predicted performance can be easily applied in an onsite scenario with detailed operational control strategies. The approach has the potential to replace traditional end-point monitoring approaches.