Chemical and Biomolecular Engineering - Theses

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    Interfacial rheology of fluid-fluid interfaces at the micro to nanoscale with AFM
    Biviano, Matthew Dominic ( 2019)
    This thesis introduces and benchmarks 2 novel AFM based techniques to characterise the interfacial rheology of polymeric fluid-fluid interfaces. We develop and benchmark a time-dependent extension of capsule compression to model oscillatory indentations, determining the interfacial rheology of capsules from thin shells to thick shells. This is first tested and benchmarked using the food-based emulsifier beta-lactoglobulin at the MCT-Oil interface. This system is then crosslinked and the behavioural changes in the capsule oscillatory rheology, bicone shear rheology and pendant drop dilational rheology are tested. This investigation has shown that there is a significant frequency dependent response of beta-lactoglobulin capsules to an oscillatory indentation in both the native and the crosslinked state. We also show that there is a large indentation depth dependence on the time dependent dwell behaviour. When comparing these behaviours to the bicone shear rheology and pendant drop dilational rheology, the magnitude of the moduli at similar frequencies differs strongly between all techniques. Comparatively, the tan(delta) between the bicone and capsule measurements exhibits similar magnitudes for both the native and crosslinked cases. An extension of the capsule technique was made to include thicker interfaces, where the thickness of the interface is more than 5% of the overall capsule radius. This model was tested with the food hydrocolloid chitosan, which in acetate buffer concentrations above 0.3M makes particulates and becomes surface active, forming a thick film at the interface. We determine the properties of this film by assessing the film mechanically and visually. To mechanically characterise the film, we utilize bicone interfacial shear rheology, and both linear and oscillatory capsule compression. The film exhibits characteristic particle like behaviours and rapidly heals when broken in shear. Similar self-healing characteristics are then observed in the linear capsule compression, and the oscillatory capsule compression shows similar moduli and behaviour to that observed by the bicone. The visual characterisation was done with in-situ aqueous imaging and dry Langmuir-Blodgett imaging of a transferred film, where a thick, continuous, particulate film is observed. We continue to push the lengthscale of interfacial rheology by developing a novel method of observing the interfacial rheology at a fluid/fluid interface. This method consists of placing a cantilever tip at the interface between two fluids, laterally translating the tip and observing the torsional force on the cantilever. This force can then be transformed into an interfacial viscosity with the knowledge of the diameter of the tip at the interface. We apply this technique to two types of interfaces, linear polymeric interfaces and a globular protein interface. At the air/water interface, we test the linear polymers PEO and PSS, where we observe that shorter chain length PSS shows a reduction in the interfacial viscosity. beta-lactoglobulin is observed over time at the decane/water interface, where the interfacial viscosity increases over time, and non-linear strain dependent behaviour is observed.
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    Metal-phenolic assembly for engineering multifunctional materials: Beyond hard templates
    Lin, Gan ( 2019)
    Metal-phenolic coordination assembly for the fabrication of multifunctional materials for diverse applications, including catalysis, pharmaceutical, nanomedicine and sensing, has attracted much attention in recent years. However, the bulk of the literature of metal-phenolic materials has been focused on the assembly on hard templates using a single ligand. There is a scope in advancing the field by investigating the versatility of the assembly system in terms of different phenolic ligands, templates and assembly techniques, which could result in novel multifunctional materials. In this thesis, different assembly techniques, particularly the use of hard and soft templates and self-assembly, were explored to create metal-phenolic materials, including thin films and particles. Metal-phenolic assembly on traditional hard templates but using a complex multicomponent phenolic mixture was first investigated (Chapter 3). The metal–phenolic assembly exhibits selective properties in a series of complex multicomponent systems (including crude plant extracts), in which metal ions (FeIII) selectively assembles with low abundant but multivalent phenolic compounds (e.g., myricetrin and quercetrin) to form thin films. This selective property was independent of the substrate properties (e.g., size, morphologies and surface charge) and the resultant metal-phenolic films demonstrated promising antioxidant properties. In Chapter 4, the transition from hard templates to microemulsions (soft) templates is described. Here, pH-sensitive poly(ethylene glycol) nanoparticles with tunable sizes and morphologies were synthesized by adjusting metal-phenolic crosslinking within the microemulsions. In Chapter 5, a template-free assembly technique was also explored to create metal-phenolic particles, which can sense and swim towards an external light source with the velocity tunable by light intensity. Nuclear magnetic resonance, confocal Raman microscopy and quantum mechanics calculations provided insight of the mechanism of light-induced movement of the metal-phenolic particles. Altogether, the metal-phenolic materials engineered through different assembly approaches presented herein show well-tailored structures and unique properties for various biomedical and engineering applications.
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    A narrow pore zeolite: ZSM-25 for natural gas purification
    Zhao, Jianhua ( 2019)
    Due to both increased greenhouse gas emissions and increased natural gas demand, the development of separating CO2 and N2 from methane-rich streams (e.g. natural gas, biogas and landfill gas) has arisen worldwide research interest. Greenhouse gas emissions can be mitigated by post-combustion technology and switching the energy structure to CH4-based energy sources. Natural gas is the most significant source of CH4, which typically contains around 80%-95% CH4, less than 10% CO2 and N2, and small amounts of hydrocarbons. Hence, removing the CO2 and N2 is critical for purifying natural gas, with the respect of both increasing the energy density and preventing the corrosion of the pipeline caused by acid CO2 for transporting CH4. Adsorption-based capture of CO2 and N2 from natural gas has attracted tremendous interest owing to its economic advantages. Porous materials play very important roles in the adsorption process where the material is exposed to the gas mixture at high pressure and then desorbs at low pressure or vacuum. The significant index of evaluating a porous material is the selectivity, capacity, adsorption kinetics and regenerability. Narrow-pore zeolite (8MR zeolite) has significant potential in natural gas purification via pressure-swing adsorption (PSA), which is attributed to its pore size fitting between CO2 and CH4, and close to N2. Hence, the selectivity is relatively much higher than other zeolites (e.g. FAU, ZSM-5). However, the slow adsorption kinetics are limiting its application in the natural gas industry, and no zeolites have been found with preferential adsorbing N2 from CH4 at equilibrium, unable to effectively separate N2 from natural gas. This thesis describes the development of small-pore ZSM-25 based zeolites, and their applications in membrane separations. The study provides a rational strategy of designing ZSM-25 zeolite for effective CO2/CH4 and N2/CH4 separation in the natural gas purification industry. In this thesis, an extensive literature on 8MR zeolite for natural gas purification and their modification approaches has been sourced and analyzed in Chapter 1. Chapter 2 a Li+/ZSM-25 zeolite (LZZ) was developed via partial ion exchange of the Na+ with Li+. This exchange enabled higher CO2 capacity and adsorption kinetics due to higher pore volume and stronger affinity of CO2 with Li+, and the ultra-high CO2/CH4 selectivity remained. The CO2 isotherms showed deviation from typical Type I isotherm and 'breathing' behavior. This observation was explained by synchrotron in situ X-ray powder diffraction, demonstrating a gradual structural expansion induced by CO2. This expansion resulted in the increased CH4 admission in binary gas adsorption. This work enables the possibility of applying small-pore zeolites in natural gas purification which are kinetically-limited. Chapter 3 The Li+/ZSM-25 zeolite (LZZ) was incorporated into a commercial polymer Matrimid 5218 yielding a mixed-matrix-membrane (MMM). Li+/ZSM-25 was chosen as filler because of its fitting pore diameter between CO2 and CH4, which merely adsorbed CH4 while allowing considerable CO2 transport. The CO2/CH4 separation performance of the optimal MMMs at 5 wt% filler loading, showed higher CO2/ CH4 selectivity than that of the pristine Matrimid in both single- and mixed-gas separation. The dominant molecular sieving effect contributed to the increasing selectivity with increased pressure, showing unusual plasticization-resistance behavior. The optimized membrane (M-5) achieved ideal CO2/CH4 selectivity of 169, which surpassed the latest CO2/CH4 upper bound. Chapter 4 A new 'trapdoor' material K-ZSM-25 was designed for N2/CH4 separation by incorporating K+ as a 'door-keeping' cation. The extent of the temperature-dependent oscillations of the K+ cation regulated the accessibility of the cage, controlling the adsorption capacity of the material. There were distinguishable gate-opening temperatures (Ts) between N2 and CH4 molecules. Within this temperature range, N2 molecules had full access to the pathway into the cage, while CH4 molecules were hindered due to the blockage of K+. Both the experimental results and simulations demonstrated that K-ZSM-25 can achieve effective N2/CH4 separation at around ambient temperature with outstanding selectivity of over 30 in single gas adsorption and 5.7 in dynamic breakthrough simulation. The large N2 capacity, outstanding N2/CH4 selectivity, fast kinetics of K-ZSM-25, and it is readily regenerated ataround room temperature, all of which makes this adsorbent ideally suited to PSA-based industrial separations.
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    Nontoxic Thermochromic Materials
    Liu, Bingxin ( 2019)
    Thermochromic products have had great commercial success in the past with colour changing products, the most well-known being the American Coor’s beer can, which saw a spike in sales at its introduction. A large range of thermochromic materials and systems, such as liquid crystals, photonic crystals, gold or silver nanoparticles, leuco dye systems, polydiacetylene derivatives and aggregachromic dyes, have been developed for industrial applications. However, concerns about their safety have limited their application in the area of food packaging. Intelligent thermochromic food packaging has the potential to monitor food quality, trace thermal history and deter counterfeiting. The research presented here is aimed at the development of a nontoxic and versatile thermochromic material for the next generation of intelligent food packaging. In this work, sulfonephthalein-solvent binary systems have been demonstrated to show clear and reversible colour change which is triggered by melting or freezing of the solvent component. This system comprises of a commercially available sulfonephthalein pH indicator dye, such as chlorophenol red, bromothymol blue, bromocresol purple, bromocresol green, bromophenol blue and thymol blue, dissolved in liquid linear chain esters, acids, alcohols or water. It was determined that the colour changing mechanism is based on pi-pi stacked aggregation or disaggregation of the sulfonephthalein dyes during the phase transition of solvent. Cell toxicity tests confirmed that the sulfonephthaleins are nontoxic. With the choice of a nontoxic solvent, this binary system can form a nontoxic thermochromic material. To make this new material more broadly applicable, microencapsulation of the binary system into a practical and workable medium for applications in ink and film is necessary. Sulfonephthalein-water thermochromic system was then developed into a microcapsule form with silicone shells prepared via reaction between water and octadecyltrichlorosilane at the interface of a w/o emulsion. The obtained microcapsules also exhibited clear colour change with full reversibility and were successfully used as inks by screen printing and as additives in films. Nontoxicity of both microcapsules and films were demonstrated through cell cytotoxicity assays. To show the thermochromic performance at ambient temperatures, a hybrid polymeric sensor containing dispersed sulfonephthalein-fatty acid particles was then developed. The obtained sensor exhibited potential as a time-temperature indicator according to its irreversible thermochromic behaviour from the dissolution and diffusion of the binary system in polymeric matrix. This sensor also showed obvious and irreversible halochromic behaviour in ammonia or bio-amines vapor. These colour change functions coupled with the non-toxic nature of ingredients and ease of preparation, make these novel materials applicable for the next generation of intelligent food packaging materials.
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    Investigation into the Role of Extracellular Substances and Intrinsic Cell Characteristics on the Harvesting and Processing of Microalgal Suspensions
    de Godois Baroni, Erico ( 2019)
    Microalgae are photosynthetic microorganisms that are cultivated in dilute suspensions and have the ability of producing a range of commercially relevant intracellular products. Lipids and proteins constitute the main product classes sought, as these can be used as feedstock to produce biodiesel, nutraceutical, and pharmaceuticals. Efficiently concentrating the dilute suspensions is a prerequisite to the economical utilisation of microalgae that has posed several technical challenges. Direct centrifugation of the cultures only presents a net-positive economic balance when high value products (e.g. pharmaceuticals) are the target of the operation. For other applications (e.g. biodiesel and proteins), a two-step process of harvesting and dewatering is necessary to meet the energy requirements associated with profitable industrial operations. Chemical coagulation/flocculation followed by centrifugation have been suggested to be highly energy efficient methods to preconcentrate and dewater microalgal cultures, respectively. These operations are directly influenced by the changes that occur in the cell population and cultivation medium during growth. However, most published studies have evaluated harvesting and dewatering separate from cultivation, and the complex links between growth and concentration have not been established yet. Studies on microalgae cultivation have demonstrated that nitrogen-limited cultures tend to accumulate larger quantities of intracellular products. By removing nitrogen from the cultivation media, cell division is arrested but the cells continue to assimilate carbon dioxide, which is stored intracellularly or excreted to the medium. In this process, the cells’ volume, morphology, and biochemistry can vary extensively, as the cell population continues to assimilate carbon but can no longer reproduce. In the existing literature, however, cell size, density, and shape, as well as the release of extracellular organic matter by the cells are commonly reported either at isolated points of the growth curve or as average values. In addition, most of the experimental techniques applied (e.g. drying and chemical fixation) ultimately alter the biophysical and biochemical characteristics of the microalgal suspensions. Both the biophysical characteristics of the cell and the dissolved matter in the medium play an important role in the stability of microalgal suspensions. Thus, understanding how nitrogen availability influences on the morphology and composition of both the cell population and the organic matter released is critical to developing efficient and scalable separation and concentration processes. As the microalgal suspension is concentrated and the solids content increases, the viscosity rapidly increases, forming a slurry with a paste-like consistency that exhibits shear-thinning characteristics. This viscous behaviour arises from the formation of an interlinked network between the cells, the extracellular organic matter, the diluted salts, and any chemical coagulant/flocculant eventually present. The microstructure formed depends on the linkages between the suspended and dissolved matter and it dictates both the compressibility and permeability of the slurry, which are parameters directly associated to dewatering performance. Hence, understanding how coagulants and flocculants influence on the volume, structure, and rheology of microalgal slurries can offer tools to critically evaluate different methods used to concentrate the cell suspension. Therefore, the main objective of this thesis was to adopt a ‘horizontal approach’ to evaluate the operations of cell concentration in association with both cultivation and processing of high-solids slurries. Initially, the effect of nitrogen availability on the cell size, shape, and density of three commercially relevant microalgae (N. salina, C. vulgaris, and H. pluvialis) were investigated using methods that allowed the evaluation of the population in its native hydrated condition. Nitrogen limitation caused an increase and diversification of the cell size distribution, and a decrease and broadening of the density distributions. Gravitational settling was adopted as a proxy for the suspension stability and a model based on modifications to the Stokes’ law was developed. This model incorporated the changes in size, shape, and density of the cells, matching closely to the batch settling data considering the smallest and less dense cells in the population. These results show that well-established physical models can predict settling velocities of microalgae suspensions given accurate data of the cell properties. In addition, these findings can be extended to predictions of flocculated more concentrated microalgae harvesting systems and other oil-bearing unicellular microorganisms. To evaluate the role of nitrogen availability on the nature and distribution of the EOM released by N. salina, C. vulgaris, and H. pluvialis, samples of soluble, loosely bound, and tightly bound EOM were obtained. A combination of mechanical methods (centrifugation, ultrasonication, and heating) was used in the extraction process that aimed at maintaining the native conditions of the EOM extracts. The composition and distribution profile of the carbohydrates, organic acids, and proteins was analysed by colorimetric methods, as well as by using fluorescent excitation-emission matrices coupled to parallel factorial analysis. This is a highly sensitive technique that allows the evaluation of hydrated EOM and the identification of the five main components present. Proteins were found preferentially tightly bound to the cells. Conversely, carbohydrates and humic and fulvic acids were mostly distributed in the loosely bound and dissolved fractions of the EOM. Nitrate depletion induces an overall increase in the EOM of the cultures. Analysis of the EOM on a per-surface area based on the sizing results obtained in the initial characterisation of the biophysical properties revealed a decrease in the EOM concentration, reflecting the arrested metabolic state of the cells and a net reuptake of nitrogen. Both composition and distribution were highly species-specific and indicated that microalgae that adopt simple cell division (N. salina) generate more hydrophilic EOM, which is less problematic to concentrate. EOM concentration was then discussed in a harvesting and dewatering processing perspective, indicating that concentration operations must be adjusted to the growth-related biophysical conditions of the cells. To extend the knowledge gained on the biophysical and biochemical characteristics of the cell population to dewatering, the role of coagulation/flocculation and centrifugation on the rheology of N. salina slurries was investigated. The chemical coagulant aluminium sulphate was used, as well as the flocculants Tanfloc POP and chitosan, as to encompass a range of increasing molecular weights (MW). Consistent centrifugation was applied to dewater the slurries that were then analysed in strain sweep, viscosity, and creep experiments. A novel microtomographic method for imaging the microstructure was also developed. Results indicated that coagulants increased the compressibility and viscosity of slurries, as the concentration and MW of coagulant increased. All samples were shear-thinning, although chitosan yielded at stressed about 1 order of magnitude higher than the other treatments. Creeps tests revealed a non-monotonic behaviour that was modified by the application of shear. Structural differences in the network indicated that highly compressible slurries (chitosan) were dominated by the low-density material, suggesting that further dewatering is possible. This study provides new knowledge on the rheology and structure of biological slurries, and it has important implications for the optimisation of dewatering operations in relation to efficient downstream processing. This thesis is the first to systematically correlate cultivation, harvesting, dewatering, and processing of high-solids slurries. The theoretical, quantitative, and qualitative data presented in this body of work provide fundamental understanding of the trade-offs between lipid accumulation, particle stability, and suspension dewaterability, which are crucial to the design of efficient concentration processes. The methodology adopted in this work has also provided a framework to analyse both dilute and concentrated cell suspensions in a native, hydrated state. This thesis has also demonstrated the potential of using shear to manipulate the rheology of high-solids slurries to further the dewatering or lower the viscosity, which ultimately result in important energy savings.
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    Combining Shear and Compression: A new Perspective on Dewatering
    Höfgen, Eric ( 2019)
    In many industries competition nationally or internationally is ever developing and thereby the need for optimisation and innovation is increasing. All these industries apply solid-liquid separation or dewatering processes as an essential unit operation. Filtration is limited by the rate and extent of dewatering as a function of the solids concentration. The rate can be described by the hindered settling function, R(phi), which is inversely related to the permeability. The second limitation is the extent, which is described in compressional rheology by the compressive yield stress, py(phi) and for further cake consolidation higher pressures have to be applied. Proper characterisation is necessary and around the world several characterisation techniques and devices exist, which have to be compared. When examining different devices one can see that a variety of forces act on the material, such as compression and shear. How and in what extent does shear affect dewatering? Shear affects the dewatering by adding another mode of load application to the material and imparting a relative movement between layers. When compression and shear are independently measured, flow is achieved in shear by at least a magnitude lower than in compression. Thereby, it is of great interest to investigate the potential to influence dewatering by combining shear and compression for more effective dewatering. A novel dewatering technology called High Pressure Dewatering Rolls combines shear and compression in a unique way. With the HPDR and other dewatering devices employing shear and compression, the characterisation in combined application is necessary. A novel development is the Vane-in-a-Filter (ViaF), which combines the dead-end piston-driven filtration rig with the Vane-in-a-Cup (ViaC) method from aiming at quantifying the influence on shear for different compressional loads. In order to bridge the gap between the ViaF and the ViaC another characterisation device called Vane-under-Compressional-Loading (VuCL) is proposed by applying weights onto the suspension below the compressive yield stress at that solids concentration. With these two measurement devices combined shear and compression can be evaluated. In this thesis with publication, the solid-liquid separation theory and characterisation, the application with the HPDR and two fundamental investigative tools for combined shear and compression are going to be presented. At the start, the comparison of the different solid-liquid frameworks and the benchmark between Nutsche Filter, Compression-Permeability cell and the dead-end piston-driven filtration rig are going to be presented. Further, the High Pressure Dewatering Rolls are presented by comparing it to industrial devices on a solids concentration basis and thereby assessing this novel dewatering device combining shear and compression with an applied vacuum. This work leads to the question on how combined shear and compression can be characterised and the Vane-in-a-Filter is presented with experimental results to close this gap. Thereby, this work presents theory, industrial application and a fundamental investigation in dewatering and combined shear and compression.
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    The use of Membrane Technology for Vegetable Oil processing with Green Solvents
    Abdellah, Mohamed Hussein Ali ( 2019)
    Utilisation of n-hexane for the extraction of vegetable oil from oilseeds presents growing concerns for the environment and human health. In spite of its high miscibility with the vegetable oil constituents, low boiling point and low heat of vaporisation, n-hexane has been recently classified as carcinogenic, mutagenic and reprotoxic solvent. As a consequence, the industrial usage of hexane, particularly in food-related industries, is expected to decline. Bioderived solvents such as terpenes, which include d-limonene, p-cymene and alpha-pinene, represent promising alternatives to n-hexane. Although terpenes show high solubility with vegetable oils, the recovery of these solvents from vegetable oil mixtures by conventional evaporation is infeasible due to their high boiling points and latent heat of vaporisation. Membrane separation technology, such as solvent resistant nanofiltration represents a potential alternative to heat intensive conventional evaporation. In this thesis, the sorption kinetics of pure solvents and their corresponding canola oil solution (0-100 wt% oil) in free standing polydimethylsiloxane (PDMS) films was studied. The PDMS films were prepared from 50 wt% PDMS solution in n-heptane with a polymer to cross-linker ratio of 2:1 (w/w). The highest sorption levels were observed with pinene followed by limonene and cymene while sorption was negligible in pure oil. Pure solvent flux curves were not linear with transmembrane pressure, but this was shown to be consistent with Fickian diffusion behaviour for highly swollen polymers. In the case of oil/solvent mixtures, the total sorption level decreased exponentially with increasing the oil content in the mixture which is explained by the decrease in the degree of membrane swelling due to the decrease of solvent activity. However, the partial oil uptake from the mixture was higher than that of the pure oil which is also attributed to the effects of membrane swelling induced by the solvents. A binary component Flory-Huggins model was used to calculate the interaction parameter between each penetrant and PDMS while a multicomponent Flory-Huggins model was used to fit the sorption isotherms of the oil and the solvent from the binary mixtures. The performance of laboratory-made polydimethylsiloxane/polyacrylonitrile (PDMS/PAN) composite membranes was studied for the recovery of terpenes from their binary mixture with canola oil. The membranes were prepared by the solution casting method from 7 wt% PDMS solution in n-heptane with a polymer to cross-linker ratio of 2:1 (w/w). The effects of transmembrane pressure (5–30 bar), feed temperature (25–40 deg. C) and feed concentration (10–30 wt% oil) on the membrane performance were investigated in terms of permeate flux and oil retention. In compliance with the observed sorption levels, pure limonene and its oil solutions showed the highest permeate flux followed by cymene then pinene. For the different oil/solvent solutions, the oil retention was comparable and increased with transmembrane pressure to more than 80 % beyond 20 bar. This increase in oil retention is attributed to the increase in solvent flux with pressure whereas the oil flux almost remained unchanged. Increasing the feed temperature resulted in an improvement in the permeate flux and a slight deterioration in membrane selectivity. The effect of cross-flow velocity on the membrane performance was also investigated and used to develop a mass transfer correlation for the boundary layer. Moreover, a mathematical model originating from the Maxwell-Stefan diffusion model in combination with the multicomponent Flory-Huggins solubility model were used to predict the flux of the solvent and the oil which was in a good agreement with the experimental data. The long-term stability of the PDMS/PAN membrane in the different oil/solvent mixtures was investigated. The membrane showed excellent stability with no observed change in performance over a period of 1 year. Furthermore, the degumming of crude canola oil diluted with terpenes by ultrafiltration was investigated. The performance of polyethersulfone, polysulfone and ceramic ultrafiltration membranes was studied at a pressure of 3 bar, feed temperature of 25–40 deg. C and feed concentration of 10–30 wt% oil. The membrane performance was evaluated in terms of phospholipid retention, oil retention and permeate flux. The polymeric membranes used in this thesis proved inadequate due to irreversible swelling of the polymer structure. The average phospholipid retention of the ceramic membrane (MWCO 5 kDa) was 95+/-2% which corresponds to a residual phosphorus content of less than 30 ppm which is consistent with conventional degumming. Conversely, the average oil retention was 16+/-3% which could be problematic in the industrial application. The highest permeate flux was observed with hexane then cymene, limonene and pinene/oil solutions. Membranes experienced fouling during operation indicated by the gradual flux deterioration with time. However, washing with n-hexane at 40 deg. C was efficient to recover the ceramic membrane performance.
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    Utilisation of glycerol as a carbon source for mixotrophic growth and lipid accumulation in marine microalgae
    Poddar, Nature ( 2019)
    Microalgal-based biomass is a promising feedstock for biofuel, chemicals and food production to meet the increasing demands for sustainable energy and material. Microalgae are photosynthetic microorganisms that use sunlight energy to power the assimilation of atmospheric CO2 into algal biomass, via photoautotrophy. Achieving high biomass concentrations and lipid productivities are some of the major challenges for large scale phototrophic cultivation. This thesis explored mixotrophic cultivation, in which an organic carbon source (glycerol) is used to supplement energy and carbon supplies to address the limitations of the conventional photoautotrophic cultivation. Mixotrophy is the simultaneous assimilation of organic carbon and CO2, with the organic carbon being used as both a carbon and energy source. With a focus on reducing costs, the present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation. This thesis provides the first descriptive analysis of the assimilation of glycerol in microalgal metabolism under different growth parameters including the availability of light and nitrogen, and the type of nitrogen provided. Nitrogen starvation is a well-known method to induce triacylglyceride (TAG) accumulation in microalgae, with the TAG being an ideal feedstock for biofuel or food oils. However, application of nitrogen-starvation strategies for TAG accumulation under mixotrophic conditions remains limited. In this study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrogen availability. Under nitrogen (nitrate) sufficient conditions, the biomass productivities of Nannochloropsis salina and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol, thereby indicating the importance of the combined effects of carbon and nitrogen. Mixotrophy is a well-studied growth regime for algal cultivation. However, the proliferation and potential role of heterotrophic bacteria in the presence of organic carbon has largely been neglected in the existing literature. The current study addresses these gaps by analysing the abundance and role of bacteria during mixotrophic growth. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to Alpha and Gamma subclasses of Proteobacteria (Paracoccus sp., and Marinobacter alkaliphilus and Marinobacter lipolyticus, respectively) in mixotrophic cultures of N. salina. Among these strains, M. alkaliphilus was shown to have aerobic denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor, even in the presence of oxygen derived via algal photosynthesis. This provided an explanation for the utilisation of nitrate and glycerol that was observed in mixotrophic and heterotrophic cultures despite limited microalgal growth. While much research has been devoted to understanding the role of nitrogen on microalgal growth, there has so far been relatively little consideration of the effect of nitrogen source on the diversity of bacteria in algal cultures. This study compared the difference between two nitrogen sources (ammonium and nitrate) on the growth of both N. salina and the associated bacteria. The performance of N. salina and the abundance, composition, and profile of bacterial groups were compared between axenic (ampicillin-containing) and non-axenic (ampicillin-free) photoautotrophic and mixotrophic cultures. The productivity of N. salina was higher with ammonium than with nitrate, with ammonium being assimilated faster than nitrate. 16S rRNA sequencing revealed the bacterial groups in the ampicillin-free cultures to include Alphaproteobacteria (predominantly Beijerinckiaceae), Gammaproteobacteria (Pseudomonadaceae and Alteromonadaceae) and Cytophagia (Cyclobacteriaceae). Pseudomonadaceae proliferated in the ammonium cultures, Alteromonadaceae in the nitrate cultures, while Beijerinckiaceae was prevalent in both ammonium and nitrate cultures. Although the presence of bacteria reduced the overall productivity of N. salina, the abundance and type of bacteria did not appear to be directly correlated with the extent of algal growth impairment. Importantly, it was revealed that both nitrate and glycerol are wasted by bacterial denitrification. As a chemically reduced form of nitrogen, ammonium did not have this problem, meaning it allows better utilisation of both nutrients for algae production rather than bacterial energy generation. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for mixotrophic algal cultivation regimes. Finally, assimilation of glycerol into the acyl chains of TAG and membrane lipids in N. salina grown mixotrophically under nitrate replete and deplete conditions was investigated using metabolomic and mass-spectrometry approaches. N. salina consumed 78% and 13% of glycerol under nitrate replete and deplete conditions, respectively. Consequently, the absolute value of total 13C incorporation was 10.6 times higher in nitrate replete than deplete conditions. However, the relative abundance of 13C incorporation was found to be higher on saturated and monounsaturated acyl chains of TAG and lower on polyunsaturated acyl chains. It was initially hypothesised in this study that glycerol would directly enhance TAG synthesis by providing a full-formed glycerol backbone on which acyl chains could be attached via the Kennedy pathway. While a disproportionate amount of fully (triple 13C) labelled lipid-associated glycerol backbones suggested that intact glycerol molecules as well as individual 13C carbon from central metabolism were used as lipid backbones, only a very low proportion of the glycerol was assimilated this way. Under nitrate replete conditions, while 43% of the carbon in the glycerol was assimilated into lipids, most of this went in membrane lipids rather than TAG. Under nitrate deplete conditions, only a small proportion (13%) of the very limited amount of glycerol that was consumed was fixed into the lipids. The knowledge and new understanding of the importance of glycerol, nitrogen availability and source, and bacterial groups that was developed through this study will be beneficial for the development of efficient mixotrophic cultivation regimes at large scale.
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    Engineering glycogen-siRNA constructs with bioactive properties
    Wojnilowicz, Marcin ( 2019)
    RNA therapeutics, such as small interfering RNA (siRNA), have great potential for the treatment of inherited and acquired diseases that are not curable with conventional methods. The delivery of new genetic material into cells provides an opportunity to alter the expression of malfunctioning genes. However, siRNA is a hydrophilic and negatively charged molecule, which cannot easily cross biological membranes and is susceptible to degradation by nucleases present in biological fluids. Therefore, siRNA therapeutics require carriers that can effectively deliver their cargo into target cells. Early formulations for siRNA delivery involved systems based on viral vectors, lipid-based nanoparticles and cationic polymers. However, these formulations often displayed high toxicity, immunogenicity, instability in biological media, inability to penetrate tissue, and/or rapid clearance from the blood stream. Fine control over carrier size and surface properties, use of simplified and reproducible synthesis approaches, and deeper understanding of the interactions between siRNA-nanoconstructs in extra- and intracellular environment can potentially improve the engineering of new carriers. In this thesis, influence of the structural properties of soft glycogen nanoparticles on the formation of siRNA constructs and their delivery in a complex biological environment were investigated. Glycogen is a hyper-branched glucose bio-polymer of nanometer size that may be isolated from various animal tissues or plants. It is composed of repeating units of glucose connected by linear α-D-(1−4) glycosidic linkages with α-D-(1-6) branching. In this work, the properties of soft glycogen nanoparticles were tailored for the engineering of glycogen-siRNA constructs. These constructs were carefully designed to efficiently penetrate 3D multicellular tumour spheroids and exert a significant gene silencing effect. Obtained results suggest that 20 nm glycogen nanoparticles are optimal for complexation and efficient delivery of siRNA. The chemical composition, surface charge, and size of glycogen-siRNA constructs were finely controlled to minimize interactions with serum proteins which influence the stability and integrity of the glycogen-siRNA constructs. pH-sensitive moieties were introduced within the construct to enhance early endosomal escape. Using single molecule super-resolution microscopy, we demonstrate that the architecture of glycogen-siRNA constructs and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. The interactions of glycogen-siRNA constructs with immune cells were also investigated, suggesting that glycogen-siRNA constructs may be cleared from the blood stream by mononuclear phagocytic system, but can still successfully deliver the therapeutic cargo.
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    Nanoengineering surface wettability via metal-phenolic networks
    Pan, Shuaijun ( 2019)
    Surface engineering is extensively involved in many industrial processes as well as diverse research applications including the engineering of catalysts, nanoparticles, coatings, membranes, gels, and other materials. In general, the purpose of surface engineering is to alter the physical or chemical surface properties on the molecular, nano-, micro-, or macroscale in order to target desired applications. Surface wetting, a ubiquitous natural interfacial phenomenon, is one of the most important yet least understood surface properties useful for addressing a broad range of practical and scientific issues. Surface wetting is also important for various global challenges such as the emerging energy and environment crises, and various health and safety concerns. One particularly “hot” scientific topic surrounding surface wetting is engineering coatings to render surfaces with tailored wettability, including coatings that are hydrophilic, hydrophobic, oleophobic, responsive, or versatile. Metal-phenolic networks, coordination assemblies between metal ions and polyphenols, are emerging conformal coating materials useful for versatile surface engineering, including the engineering of nanomaterials and bio-interfaces. Polyphenols, which are abundant in natural sources as well as synthetic chemicals, have outstanding physicochemical properties besides metal chelation, such as reactive chemical groups, special interfacial interactions, and controllable bioactivity, and thus provide vast potentials in the field of advanced surface modifications. However, the potential of polyphenols in surface wetting has rarely been investigated, leaving the fundamental understanding necessary for advanced surface design and surface wetting largely unclear and incomplete. Therefore, this thesis aims to provide an overview of the wetting potential of metal-phenolic networks and to provide fundamental understandings for engineering advanced surface wetting. Specifically, this thesis investigates engineering surfaces on the levels of chemical structure, coating composition, and substrate hierarchy. Finally, some emerging applications for metal-phenolic networks with tailored surface wetting are presented. The scope of this thesis is the engineering and exploitation of the surface wetting of metal-phenolic networks. The wetting fundamentals of metal-phenolic networks are first explored through the systematic study of coatings prepared from a wide range of polyphenolic ligands and a collection of metal ions on diverse substrates utilizing a series of coating methods. The intrinsic wetting properties, together with the active surface nature, were then successfully exploited for various applications including catalysis, oil-water separations, air filtration, and self-cleaning. The toolbox applicable building blocks for making metal-phenolic materials was enlarged by introducing the concept of host-guest chemistry—host functionality is incorporated into metal-phenolic networks where guest molecules can specifically bind with the host motifs within the surface coating. In addition to the facile control over surface wetting and specific binding, the host-guest metal-phenolic networks can also potentially help tackle incompatibility problems encountered in the design of materials for advanced interfacial interactions. Finally, dynamic metal-phenolic networks capable of adaptively interacting with a range of liquids were engineered. The resultant adaptive surface wetting, along with the broad wetting potentials of the metal-phenolic networks, is expected to contribute to the engineering of advanced surface coatings and find applications in other fields requiring tunable interactions.