Chemical and Biomolecular Engineering - Theses
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Nontoxic Thermochromic Materials
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.
Investigation into the Role of Extracellular Substances and Intrinsic Cell Characteristics on the Harvesting and Processing of Microalgal Suspensions
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.
Combining Shear and Compression: A new Perspective on Dewatering
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.
The use of Membrane Technology for Vegetable Oil processing with Green Solvents
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.
Utilisation of glycerol as a carbon source for mixotrophic growth and lipid accumulation in marine microalgae
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.
Engineering glycogen-siRNA constructs with bioactive properties
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.
Nanoengineering surface wettability via metal-phenolic networks
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.
Harnessing the power of the Fenton reaction in RAFT polymerization technique
Polymer scientists have been attempting to synthesize tailored polymers with predetermined molecular weight, composition, architecture, and molecular weight distribution by moving from traditional free radical polymerization (FRP) to reversible deactivation radical polymerization (RDRP) techniques, i.e. atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and nitroxide-mediated polymerization (NMP). Redox-activated FRP initiated by a classical chemical reaction (i.e., Fenton reaction) has been extensively used for the synthesis of different monomers and fabrication of various polymeric materials. Fenton reaction describes the reduction process of hydrogen peroxide (H2O2) by ferrous ions (Fe2+), generating highly reactive hydroxyl radicals. Despite its unique features such as high reaction speed and cheap reagents, Fenton chemistry was not employed for the initiation of RDRP methods. This thesis proposes the application of the Fenton reaction for initiating the RAFT technique at ambient conditions. The presentation is organized by the manipulation of H2O2 and Fe2+ sources and type of monomers. This work leads to the synthesis of well-controlled linear homo- and co-polymers with different polymer chain lengths. In the 1st part, the Fenton reaction was introduced to the RAFT process for the first time, termed as Fenton-RAFT polymerization, as an “on-demand” chain growth method. The ultra-fast Fenton–RAFT technique resulted in well-defined hydrophilic polymers with high monomer conversions (~ 75%) within 1 min at room temperature. The study on the polymerization rate and polymers’ characteristics in the presence of air, showed oxygen-tolerance of the Fenton-RAFT system with good control over polymers’ size. The 2nd part of this study directly addressed the drawbacks of Fenton-RAFT process (i.e., non-affordable full conversions and presence of metal ions) by replacing synthetic H2O2 and inorganic source of Fe2+ (i.e., ammonium ferrous sulphate) with two proteins, i.e. glucose oxidase (GOx) and hemoglobin (Hb). Biologically activated Fenton-RAFT polymerization was termed as Bio-Fenton-RAFT. Since Bio-Fenton-RAFT successfully led to the synthesis of well-defined polymers with full conversion values in either water and biological media, a RAFT polymerization catalyzed by real red blood cells was attempted. The Bio-Fenton-RAFT and blood-catalyzed Fenton-RAFT systems showed excellent tolerance towards oxygen and control over polymer chain lengths. In the 3rd part, we took advantage of the capability of the catalyst system (i.e., GOx/Hb) via the Bio-Fenton-RAFT process in supplying initiating radicals to make ultra-high molecular weight (UHMW) polymers. This technique led to the synthesis of unprecedented large, well-controlled UHMW polymers with a molecular weight as 20 × 106 g mol-1. The amount of GOx-generated H2O2 was detected to be ~ 3 mM in reaction solution within 2 h. To reduce the reaction time, Hb was replaced with ammonium ferrous sulphate. Synthetic H2O2 was carefully and gradually injected and added to the reaction solution by using a syringe pump, leading to well-defined UHMW polymers after 2 h. Such controlled production of initiating radicals offers unique access to predefined UHMW polymer materials via other RAFT processes. In the 4th part, I attempted to address the challenge (i.e., catalyst residuals) I faced in all previous Fenton-RAFT processes. To do so, I developed heterogeneously catalyzed Fenton-RAFT technique by using Fe2+ metal-organic framework (MOF) particles, termed as MOF-Fenton-RAFT. In this methodology, synthetic H2O2 was used, and ammonium ferrous sulphate, commercial Hb, and native Hb were replaced with heterogeneous catalysts, Fe2+ MOF particles. The obtained results demonstrated that MOF-Fenton-RAFT is pH-dependent, and acid-bearing RAFT agents play an important role in the rate of RAFT polymerization.
Immobilised solvent systems for carbon capture
Microencapsulated solvents (MECS) are a novel approach to carbon capture, with the potential to reduce unit operation volumes by 1-2 orders of magnitude, and to allow a wide range of solvents to be contacted with a flue gas stream in a practical way. In this technology, small droplets of a solvent which selectivity absorbs CO2 are encapsulated inside thin polymer shells, which immobilise the liquid but allow CO2 to easily pass through. The capsules have diameters of 100 - 600 μm, which corresponds to a surface area 1 - 2 orders of magnitude greater than the specific area of a fluid flowing over random or structured packing in a traditional absorber. A fluidised bed containing fine MECS could plausibly be over 10 times smaller than a traditional absorber, and may allow solvents with slow absorption kinetics to capture CO2 in a practical way. In Chapter 3, it is found that microencapsulation is unlikely to affect the gas flux for concentrated chemical solvents, while for physical solvents it may increase the gas flux under some circumstances, as the reduction in spatial scales increases concentration gradients within the fluid. Overall, microencapsulation may increase the gas absorption rate by an order of magnitude for chemical solvents, and by 2 orders of magnitude for physical solvents. In Chapter 4, a novel material for carbon capture, Solvent Impregnated Polymers (SIPs), is proposed. SIPs have many of the favourable properties of MECS, but are more scalable to manufacture. SIPs were manufactured containing various solvents for CCS, including K2CO3 solutions, an ionic liquid, and a Nanoparticle Organic Hybrid Material. A validated mass transfer model found that SIPs could increase the gas flux by a factor of 2-4 when immobilising solvents in the pseudo-first order reaction regime, and by 1-2 orders of magnitude for solvents in the instantaneous reaction regime. A 50-fold increase in flux was experimentally observed in a SIP containing a Nanoparticle Organic Hybrid Material. If such an increase in flux were combined with an increase in surface area of 1-2 orders of magnitude, this could plausibly lead to a 3-4 order of magnitude increase in the specific gas absorption rate, which may enable slower solvents to be used in a practical way. In Chapter 5, the energy savings which SIPs could provide were analysed. Because of the remarkable efficiency of modern amine-based CCS processes, it is found that even SIPs or MECS containing `optimal' solvents are unlikely to provide significant energy savings, especially if steam regeneration is used. On the other hand, it is possible that SIPs or MECS containing solvents which may be regenerated using low-grade waste- or electrically-generated heat could be used in novel processes at reduced cost. Furthermore, it is found that an optimal solvent for CCS requires both a reasonably large enthalpy of absorption balanced by a sufficiently large entropy of absorption. In the development of novel solvents for CCS, the significance of the entropy of absorption has largely been ignored, and this should be considered more carefully into the future.
Bio-nano interactions of metal-phenolic networks: the role of metals
Metal-phenolic networks (MPNs) hold great promise for the fabrication of multifunctional hybrid materials owing to their versatile and tunable nature. In particular, the beneficial combination of both organic and inorganic components makes them highly interesting systems for a range of applications. Because of the ease of changing the metal ions and ligands, they are highly interesting materials for drug delivery, cell targeting, medical imaging, and catalysis. Metal ions are known to bind to a variety of biomolecules, for example amyloid beta peptides and antibodies. However, to date, the bio-nano interactions of MPNs are not fully understood, especially for the role of metals. In this PhD research, bio-nano interactions of MPN- coated gold nanoparticles (AuNP@MPNs) with different biomolecules were studied to reveal the role of metals. In the first part of this PhD research, the potential of AuNP@MPNs for amyloid fibril inhibition was investigated. Metal ions and polyphenols have been demonstrated to separately play an important role in the amyloid fibril progression and in the inhibition of fibril formation, respectively, and therefore in combination should have synergistic effects. Numerous diseases, such as Alzheimer’s disease and Type II Diabetes, are potentially associated with the formation of amyloid fibrils. In this systematic study, metal ions were varied in the MPN system, with Co-TA (cobalt-tannic acid)-coated AuNPs showing the highest inhibition ability. Molecular dynamics simulations and quantum mechanics calculations suggested that the geometry of the exposed cobalt coordination site in the cobalt-tannic acid networks facilitated its favorable interactions with histidine and methionine residues in the amyloid beta peptides. Like amyloid fibrils, antibodies can interact with transition metals (e.g., CoII, NiII, CuII, ZnII) via the histidine-rich domain at Fc region in an oriented manner. In the second part of this PhD research, AuNP@MPNs were modified with antibodies by adsorption and their targeting abilities were studied. Similar antibody loading levels were observed for all AuNP@MPNs with different metals. However, the Co-TA coated AuNPs adsorbed with antibodies again showed a different behavior compared to the other metals. It possessed improved targeting towards both antigens and cells by inducing the potential orientation (conformation) change of the adsorbed antibodies, which further confirmed the unique property of cobalt in the bio-nano interactions of MPNs. The third part of this PhD research further investigated the bio-nano interactions of AuNP@MPNs in the complex protein system – human serum. As tannic acid might dominate the bio-nano interactions, the effects of different ligands were examined along with the effects of the different metals. It was found that the protein corona can reduce the cell association of all AuNPs investigated. The amount and composition of corona proteins were evaluated by both SDS-PAGE and LC-MS/MS. MPNs with tannic acid as the phenolic ligand showed no significant difference with varied metals in both corona protein content and cell association. However, MPNs with gallic acid as the ligand showed that FeIII and ZnII exhibited different corona protein content and cell association compared to other metals. These findings suggested that bulky tannic acid may dominate the adsorption of biomolecules while cobalt can contribute to the conformation of biomolecules by coordination. Taken together, this research provides a fundamental understanding of MPNs for future bio-nano related applications.
Phage treatment of filamentous bacteria in activated sludge
The activated sludge process is one of the most commonly used wastewater treatment processes, which consists of a biochemical stage (aeration basin) and physical separation (secondary clarifier). The successful operation of this process relies on effective separation of the biomass from the treated wastewater. Under certain operating conditions, however, excessive growth of filamentous bacteria can cause sludge bulking and foaming, which limits solid-liquid separation, negatively impacting process performance. Currently, no effective treatment method exists that selectively controls the abundance of troublesome filamentous bacteria without adversely impacting the microbial ecology. The application of phages, viruses that infect and lyse bacteria, is potentially a target-specific and sustainable biocontrol method that could combat operational problems associated with the overabundance of filamentous bacteria; a strategy that has received increased interest over the past decade. Successful phage application, however, requires a better understanding of the influence of filamentous bacteria on the physical characteristics of the activated sludge flocs that impact solid-liquid separation and floc stability. Additionally, a better understanding of the adsorption of phages onto flocs and diffusion of phage within these structures is required, as these two factors will impact the efficacy and dynamics of phage treatment in the activated sludge process. The main objective of this research was therefore to investigate these factors and assess their influence on the application of phages in order to further develop phage as a potential treatment method for problematic filamentous bacteria associated with both foaming and sludge bulking. In this project, a semi-automated image analysis method was developed and applied to quantify the abundance of filamentous bacteria in activated sludge. The length of protruding filaments was normalised to the floc area, which gives a relative measure of filament abundance. It was shown that a higher abundance of filaments enhanced the resistance of activated sludge flocs to shear-induced breakup, which is in agreement with the filamentous backbone theory. Flocs with a higher abundance of filamentous bacteria, however, also had an adverse impact on solid-liquid separation, increasing the resistance to settling, as quantified by the hindered settling function at solid concentrations below the gel point. An increased abundance of filaments also caused poorer sludge compactability, as reflected by lower gel point concentrations. From these observations it is evident that a balance between the filamentous and floc-forming bacteria is desired. Ideally, the abundance of filamentous bacteria should be sufficiently high to enhance floc stability but sufficiently low to prevent poor performance of solid-liquid separation processes. Importantly, this work refocuses the attention on the vital role of filamentous bacteria in floc stability, which is in agreement with the filamentous backbone theory. The adsorption of phages to activated sludge flocs is an important consideration for phage treatment of filamentous bacteria that affects both the likelihood of individual infection and the dynamics of phage infection in the system but has not been studied to date. Systematic adsorption experiments were performed under controlled conditions using model alginate surfaces that resemble the extracellular polymeric substances (EPS) of activated sludge flocs. Theoretical interaction energies were calculated and compared to experimental deposition data to determine the dominant interactions between phage particles and the alginate surface. These calculations sought to examine both electrostatic and hydrophobic interactions. An increase in the concentration of counter ions resulted in a higher degree of electrostatic screening and enhanced phage deposition. The relatively high hydrophobicity of phage GTE6 also resulted in a significant contribution of hydrophobic attraction to the overall interaction energy. The attractive hydrophobic interaction counters the repulsive electrostatic interaction and results in more favourable conditions for phage adsorption. The adsorption of phage GTE6 to activated sludge flocs was also investigated by means of batch adsorption experiments. Kinetic and isotherm parameters of adsorption were determined, providing valuable insights regarding the application of phages to the activated sludge process. These parameters should be incorporated into dynamic models of phage treatment in the activated sludge process to further evaluate the impact of adsorption on the dynamics and efficacy of phage treatment for filamentous bacteria. It is important to take these effects into account when dosing strategies are developed for application at plant level. The diffusion of phages into activated sludge flocs will also influence the effectiveness of phage treatment. Model systems consisting of alginate beads with immobilized Gordonia terrae were developed to demonstrate the influence of the matrix structure and the potential protective effect provided by the EPS of activated sludge flocs against phage infection. Alginate beads prepared by internal gelation were used as a model system to represent activated sludge flocs with a loose EPS matrix, whereas external gelation was used to represent flocs with a relatively dense EPS matrix or compact EPS layer surrounding the floc. The spread of infection and subsequent lysis of embedded host bacteria were measured by means of confocal scanning laser microscopy and fluorescent viability staining. The matrix structure of alginate beads prepared by internal gelation permitted diffusion of phage GTE6 and subsequent infection of embedded host bacteria. The presence of the alginate matrix resulted in slower diffusion of phages and subsequently slower infection kinetics compared to dispersed bacteria in the absence of a matrix structure. A denser matrix at the periphery of alginate beads, prepared by external gelation, prevented diffusion of phage GTE6 into the alginate beads and therefore protected the embedded host bacteria against phage infection. The results from these different systems, which potentially represent activated sludge flocs with low and high solids retention times, suggest that phage diffusion into flocs may be restricted by the denser EPS matrix of flocs associated with longer retention times. Under such conditions, the filamentous backbone within the confines of the flocs will be protected against phage infection, while free-floating and protruding filamentous bacteria, which are responsible for foaming or bulking when abundant, are readily accessible to phages for infection and subsequent lysis. This finding impacts the dynamics of phage infection and should be considered in model development and future implementation plans. Overall, the knowledge gained through this thesis provides a better understanding of practical aspects that will impact the application of phages to control filamentous bacteria in the activated sludge process. This research confirms that filamentous bacteria are necessary to enhance floc stability but if excessively abundant will adversely affect solid-liquid separation. Furthermore, this research has improved our understanding of phage adsorption to EPS and activated sludge flocs, factors that will impact the dynamics and effectiveness of phage treatment in the activated sludge process. The model systems applied in this work illustrated the impact of the EPS matrix structure on the accessibility of filamentous bacteria within the confines of activated sludge flocs, which will have a significant influence on the efficacy of phage treatment and impact process performance. This improved understanding will assist modelling of the system and guide the development of phage dosing strategies to reduce the risks associated with the implementation of phage treatment at plant level. The toolsets and framework provided in this thesis should be applied in further application studies. The semi-automated image analysis method can be used for routine quantitative assessment of filament abundance at plant level, which reduces operator bias and is useful for improved process monitoring and research purposes. Additionally, a useful framework was developed for phage application studies in which the 3-dimensional growth of bacteria and spread of phage infection in alginate matrices can be investigated by means of confocal laser scanning microscopy. This technique will be valuable in the broader field of phage biology, for example in the application of phage to other flocculated or biofilm systems.
Simulating the behaviour of skim-milk during ultrafiltration
Many commercial dairy products such as cheese and concentrated milk proteins are produced via ultrafiltration of skim-milk. During skim-milk ultrafiltration, a permeate stream comprising lactose, water and other minerals is removed from the system leaving behind the retentate, rich in whey proteins and casein micelles. The retained proteins result in a concentration boundary layer near the membrane. Solids build-up within this boundary layer results in resistance to the permeate flow in addition to the membrane resistance, causing a flux decline. Solids build-up also increases the contamination risk in the retained product. In order to overcome these problems, periodic cleaning of the membrane is performed which increases the overall operational cost. Numerical models that predict the behaviour of a filtration process have gained importance in the development of membrane modules and optimizing operating conditions. While there have been numerous studies based on the single-phase models, for the non-dilute volume fractions relevant to ultrafiltration applications, this framework lacks rigorous theoretical justification. A multiphase framework is better equipped to model solid-solid and solid-fluid interactions. In this thesis, a mixture model based on the multiphase framework is developed to predict the behaviour of dairy filtration. In this model, continuity and momentum equations are solved for the mixture, in addition to a solid phase continuity equation that employs a relative velocity between the phases, calculated from the differences in forces acting on each phase. This thesis details the implementation of the mixture model for typical filtration channels with large aspect ratios via geometric scaling. Mathematical similarity is achieved between the actual and the scaled filtration domains. The mixture model is then used to simulate the ultrafiltration of whey proteins in both dead-end and crossflow filtration modes. Simulations that employ hard-sphere models for the osmotic pressure and drag coefficient produce results that are consistent with relevant dead-end and crossflow filtration experiments, with the use of independently evaluated parameters such as voluminosity and high-density gel pore length scale. A deformation model is then proposed to capture the particle deformation occurring during ultrafiltration in the regions with high concentrations. This model, based on the Young's modulus of the material and initial particle size, is able to predict the filtration of deformable particle systems such as soft latex and casein micelles dispersions in the dead-end mode. The largest discrepancy between simulations and experiments occurs for skim milk during crossflow ultrafiltration - possible reasons for this discrepancy are discussed and presented as avenues for future work. The mixture model and the deformation model presented in this thesis are important in developing a comprehensive model to predict the behaviour of skim-milk ultrafiltration. This model is robust and valid across a wide range of operating conditions for filtration of proteins and latex dispersions. Though the present model is capable of modelling only one dispersed phase (either whey proteins or casein micelles), the model can be extended in the future for the study of two or more dispersed phases, relevant to the multicomponent nature of real milk.