Theses - Chemical Engineering and Biotechnology

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Open Access
Experimental investigation on the effect of the combustion of oxygenated fuels on the formation of soot
Tan, Yong Ren; Tan, Yong Ren [0000-0002-8029-9027]
The transport sector is a major contributor to the global greenhouse gas and pollutant emissions, owing to the high reliance on fossil-based fuels. In light of this, the search for sustainable alternative fuels is imperative. Oxygenated fuels have been proposed as a potential solution for their sustainable nature and clean combustion properties. However, it is crucial to understand the potential pollutants (particularly soot) generated during the combustion of oxygenated fuel because of their distinct chemical composition when compared to fossil-based fuels. To have a through investigation on oxygenated fuel combustion and its impact on soot formation, three different combustion systems are examined to determine how oxygenated fuels affect soot formation in this thesis. Experimental methods including colour-ratio pyrometry, differential mobility spectrometry, Raman spectroscopy, and thermogravimetric analysis are used to characterise the effect of oxygenated fuels on soot formation. The first study focuses on the impact of blending proportions of oxygenated fuels on soot formation under laminar coflow diffusion flames. Polyoxymethylene dimethyl ethers (PODE_n) were chosen to be studied because they are one of the emerging class of oxygenated fuels for the transport industry. Up to 20% of PODE₃ was blended to ethylene to generate the flames to perform flame temperature, soot volume fraction, and particle size distribution measurements. The 5% PODE₃ blend showed a synergistic effect in the formation of soot, while higher blends reduced the soot volume fraction and average particle size. The formation of the initial benzene (and subsequently soot) was linked to the pathways by which the fuels decompose. In order to compare the differences between oxygenated fuels, three C3 oxygenated fuels - PODE₁, *iso*-propanol, and dimethyl carbonate (DMC) were studied using the same methodology as PODE₃ due to the intriguing synergistic effect observed for PODE₃. The C3 oxygenated fuels exhibited distinctly different degree of the synergistic effect on soot formation at the same blending ratio. The findings reinforce the importance in considering the fuel molecular structure in influencing fuel decomposition pathways, which in turn affect soot formation. Four oxygenated fuels - ethanol (EtOH), DMC, PODE₁ and PODE₃ - were mixed with jet fuel and investigated using wick-fed laminar diffusion flames to better understand the sooting behaviour of oxygenated fuel mixtures in a more complex chemical environment. Regardless of the type of oxygenated fuels, it was discovered that the sooting tendency (as measured in accordance with ASTM D1322) generally correlated with the soot volume fraction and particle size distribution measurements. The ability to relate data gathered using the ASTM D1322 standard for the sooting behaviour of different mixtures will be beneficial for the aviation industry upon the switch to sustainable fuels. Finally, under a compression ignition engine, the morphology and nanostructure of soot generated from the combustion of oxygenated fuels (EtOH, DMC, PODE₁ and PODE₄) with jet fuel were investigated. It was revealed that the dilution effect, combustion condition effect, and chemical effect are all possibilities in which the blending of oxygenated fuels affects the properties of soot. Particularly, the type of oxygenated species produced during the combustion of oxygenated fuels can have a significant impact on the soot properties.
Embargo
Continuous synthesis of metal halide perovskite nanoparticles with sharp and stable emission
Zhang, Kaiwen
Metal halide perovskite nanoparticles present unique and attractive optical and electronic properties, leading to promising application in optoelectronic devices such as light-emitting diodes and solar cells. The optical properties of metal halide perovskite nanoparticles are tuneable by size, therefore, synthesizing metal halide perovskite nanoparticles with size control, especially the quasi-2D nanoplatelets, are of great interest. However, the lack of deep understanding of the synthesis reaction hinders further development of the quasi-2D metal halide perovskite nanoplatelets. The lack of size control and thickness tuning impedes the colour-pure and tuneable emission obtainable from monodisperse metal halide perovskite nanoplatelets. In addition, the stability of these materials is relatively low, hence requiring further development. This thesis focuses on the controllable synthesis of caesium lead halide (CsPbX₃) nanoplatelets (NPls) with size tunability and phase purity using continuous flow reactors and the stability improvement of CsPbX₃ nanocrystals (NCs). The controllable synthesis is achieved by reactor engineering, and the importance of mass transport in the synthetic processes is revealed. The stability improvement is attained by encapsulation approach with a series of materials. First, the importance of transport phenomena on the synthesis of metal halide perovskite nanoplatelets is illustrated with CsPbBr₃ NPl synthesis reaction in batch and flow reactors. The need for fast and homogeneous mixing is demonstrated to achieve monodispersed crystals, in this case CsPbBr₃ NPls with sharp blue emission. The modularity of the synthesis method, combining fluid dynamic simulations in a collaboration, bespoke 3D flow reactors, and on-line measurements (UV-vis absorbance and photoluminescence) reveals fundamental understanding about the growth of these perovskite nanomaterials, which in turn leads to superlative size reducibility (2.2 ± 0.3 nm) and properties (sharp blue emission at 472 nm wavelength, and PLQY 24.5±0.4%). Understanding the dynamics of the synthesis in the very early stages (within the first 100-300 milliseconds) shows that mixing of the precursors plays a key role not only during the nucleation step, as previously believed, but also during the growth stage. This work also demonstrates that the flow reactors can provide a consistent and steady output capable of fulfilling the large demand of blue light emitters for a variety of applications. Second, the phase pure CsPbI₃ NPls are selectively synthesized in flow reactors, obtaining monodisperse thicknesses of 3 and 4 monolayers, of which the latter has the most suitable emission wavelength for red emitters in display applications. The synthesis study using two different reactors reveals that efficient mixing is critical for obtaining both phase pure CsPbI₃ NPls, while the fast or very fast mixing critically controls the synthesis by manipulating monomer formation to achieve selective synthesis. More importantly, the comparative studies illustrated fast growth of CsPbI₃ NPls under room temperature synthesis. This work shows that different strategies are required for different phases and demonstrates the advantage of flow systems to achieve highly reproducible large-scale synthesis of materials with fast reaction rates. Last, a series of strategies of encapsulating CsPbBr₃ NCs with metal oxide are studied aiming at improving the stability of CsPbBr₃ NCs in various scenarios. The post-synthesis treatment approach targeting at SiO₂, TiO₂, AlO_x, and NiO_x shells are studied with separate reaction steps, and their respective effectiveness are evaluated against water, solvents, irradiation, and ion exchange. Progress and challenges are presented with each shell material, and the results and discussions are useful as guidance for future studies. This thesis contributes to the metal halide perovskite field of research with controllable synthesis and tuneable optical properties to enable their potential application in optoelectronic devices. The 3D reactors demonstrate unparalleled potential in continuous synthesis of metal halide perovskite materials in large scale.
Open Access
Activation of Release by Hydration in Pharmaceutical Tablets Studied by Terahertz Pulsed Imaging
Dong, Runqiao
Tablets are one of the most common pharmaceutical dosage forms. The liquid ingress into a tablet is considered to be the first, and often rate-determining, step for tablet disintegration and subsequent drug dissolution. Even for tablets that do not disintegrate (e.g. sustained release tablets), hydration of the oral solid dosage form still plays a critical role in activating the drug release. In this thesis, terahertz pulsed imaging (TPI) was used to investigate the hydration kinetics of pharmaceutical tablets in a one-dimensional liquid penetration setup. The capability of terahertz pulses to probe through, and respond to, interfaces of different structures inside a tablet and the high data acquisition rate of TPI makes it a highly suitable tool for this study. The TPI method was first demonstrated on uncoated tablets. The kinetics of liquid penetration extracted from TPI data was compared to the corresponding dissolution testing results, and the mechanistic impact of various key ingredients in the formulation was revealed. The research then focused on coated tablets where the presence of the layered structure stepped up the complexity of the hydration process. The tablet cores were made of pure microcrystalline cellulose (MCC) and a PVA-based (polyvinyl alcohol) immediate-release coating formulation was applied in the first step. Initially, vacuum compression moulding was used to apply the coating layer. Although the coatings prepared by this method were highly porous and lacked uniformity, TPI managed to quantitatively resolve multiple stages of liquid transport through coating and tablet core, and identify a discontinuity in liquid transport at the coating-core interface in contrast to the uncoated tablets. Subsequently, a spraying system was employed to apply film coating of industrial quality. Similar multi-staged liquid transport behaviour was consistently observed and a systematic approach for data processing was developed. A multiple linear regression method was used to analyse the dependency of the hydration kinetics of coating and core layer on both coating thickness (30 μm to 250 μm) and core porosity (10% to 30%). Finally, a EC-based (ethyl cellulose) sustained-release coating was studied. The slow water ingress was characterised by the amount of swelling in core monitored by TPI instead of directly tracing the water front. Various conditions triggered in a prolonged hydration process such as crack formation were identified. The subtle details of liquid penetration captured uniquely by TPI provide valuable insights into the physical steps involved in drug release upon hydration from tablets. With suitable complementary tools, TPI presents great potential for developing better understanding to aid the rational design of pharmaceutical formulations and processes.
Open Access
Development of Bioelectronic Technologies for the Investigation of Extracellular Vesicle Function and Anti-Cancer Drug Discovery
Traberg, Walther
Extracellular vesicles (EVs) are cell-secreted phospholipid bilayer-delimited particles of varying size and composition that facilitate intercellular communication. EVs play a critical role in a wide range of diseases by mediating the transfer of active biomolecules between cells, both in the vicinity of the source cell and at distant sites, to elicit a variety of phenotypic responses. Tumour-derived (T)EVs facilitate the transfer of information between tumour and non-malignant cells to initiate and drive metastasis through a variety of processes, including the epithelial-to-mesenchymal transition (EMT) and angiogenesis. As such, TEVs represent a novel therapeutic target in a field severely lacking in efficacious anti-metastasis treatments. Research into EV biology and function has seen a boom over the last decade owing to their unique features and potential clinical utility, and the demand for new and robust tools to study their functional activity has risen commensurately. However, conventional characterisation methods fail to comprehensively capture the dynamic nature of EV function, and this has obscured the true role of EVs in pathophysiological processes despite their pleiotropic functions in vitro. Furthermore, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are missing. Here, bioelectronic technologies are utilised as a promising, scalable technology for investigating the dynamics of EV function in a truly quantitative manner using both cell-free and cell-based models. Furthermore, proof of concept drug screening studies demonstrate the applications of these bioelectronic platforms to preclinical drug discovery, addressing the current lack of robust, scalable sensing technologies that facilitate facile translation to in vivo outcomes. In this work, organic bioelectronic devices were integrated with cell-free, supported lipid bilayer (SLB) models and a cell-based breast cancer metastasis model. In the case of the former, electronic devices were used to detect virus-membrane fusion, which laid the groundwork for the subsequent study on EV-membrane binding, as viruses and EVs share many similarities in form and function. SLB integration with conducting polymer coated substrates and electronic devices was assessed using optical techniques. Virus hemifusion to both synthetic and natively derived SLBs was detected electrically using electrodes and organic electrochemical transistors (OECTs). Next, a supported biomimetic stem cell membrane incorporating membrane components from human primary adipose-derived stem cells (ADSCs) was formed to monitor the binding of cancer-derived exosomes (cEXOs) to the plasma membrane and show that this binding can be blocked when an antibody to integrin β1, a component of ADSC surface, is exposed to the membrane surface prior to cEXOs. These SLB platforms used a label-free electronic readout as well as a dual capability of optical (fluorescence) readout. Lastly, the development of a functional phenotypic screening platform based on OECTs for real-time, non-invasive monitoring of TEV-induced EMT and screening of anti-metastatic drugs is reported. TEVs derived from the triple-negative breast cancer (TNBC) cell line MDA-MB-231 induced EMT in non-malignant breast epithelial cells (MCF10A) over a 9-day period, recapitulating a model of invasive ductal carcinoma metastasis. Epigenetics and immunoblot analysis showed that TEVs modulate TWIST1 protein amount but not DNA methylation level, and dual optical and electrical readouts of cell phenotype using OECTs were obtained. Further, heparin, a competitive inhibitor of cell surface receptors, was identified as an effective blocker of TEV-induced EMT, providing proof of principle that inhibitors of TEV function can be potential anti-metastatic drug candidates. Together, these results demonstrate the utility of bioelectronic platforms presented here for studying TEV function, both in a cell-based and cell-free manner, and identifying inhibitors of TEV function for drug discovery applications. In additional to being readily scalable, organic electronics facilitate facile modelling of the transient drug response using electrical measurements and are amenable to integration with highly tuneable models systems.
Open Access
Hybridising Organic Chemical and Synthetic Biological Reaction Data for Molecular Synthesis
Zhang, Chonghuan
Computer-assisted synthesis planning (CASP) accelerates the development of organic synthetic pathways of complex functional molecules. CASP tools are usually developed from organic synthetic chemistry rules or the reaction data. However, synthetic biology offers a new degree of freedom through the ability to develop new synthetic steps. In this PhD thesis, a method for hybridising conventional organic and synthetic biological reaction datasets is presented to guide synthesis planning. Part of the organic reactions from the Reaxys database was combined with the metabolic reactions from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to create a hybrid dataset. The combined dataset was used to assemble synthetic pathways from multiple building blocks to a target molecule. Route assembly was performed using reinforcement learning, which was adapted to learn the values of molecular structures in synthetic planning and to develop a policy model to propose near-optimal multistep synthetic routes from the pool of available historical reactions. To quantify the added value of synthetic-biological reaction transformations in hybrid pathways, three policy model "decision makers" were developed from the organic, biological, and hybrid reaction pools, respectively. The near-optimal synthetic pathways predicted from the three reaction pools were evaluated and compared to discuss the advantages of synthetic chemical and synthetic biological reaction hybrid decision space in optimising reaction pathways. The hybrid pathways show that biochemical transformations may allow significant gains in syntheses efficiency, but yet, there is a limited access to biochemical reaction data, which limits the opportunity to find alternatives and synergies with organic synthesis. Hence, a workflow to explore the sparse synthetic biological domain was proposed. Learned from the biocatalytic transformations of recorded reactions, feasible biosynthetic reactions were proposed to expand KEGG reaction dataset by four folds. To catalyse the novel reactions, a transformer model learned from the reaction SMILES and amino acid sequences of native enzymes to predict promiscuous enzymes for potential substrates. The proposed transformer model calibrates the feasibility of the predicted reactions and reduces the search scope for promiscuous enzymes in the pool. The hybrid pathways also show the organic reactions are noisy, with a major issue of missing reaction co-participants in the reaction records. A heuristic-based method was developed to identify the balanced reactions from reaction databases, and complete some imbalanced reactions by adding candidate molecules. A machine learning masked language model was trained to learn from the reaction SMILES sentences of these completed reactions. The model predicted missing molecules for the incomplete reactions, analogous to predicting missing words in sentences. The model was promising to predict small and middle size missing molecules for incomplete reactions. The thesis presents an idea of hybridising organic chemical and synthetic biological reactions and delivers workflows towards a cleaner and more comprehensive hybrid reaction space to guide molecular synthesis. With the thesis presented methods, the research of CASP could be accelerated, and we could aim for more sustainable and efficient molecular synthesis routes.
Embargo
Zirconium-based Metal-Organic Frameworks for Pleural Mesothelioma Therapy
Liu, Xiewen
This thesis describes the experimental studies to improve the treatment of malignant pleural mesothelioma, an incurable cancer with a survival time of only one year after diagnosis. Mesothelioma is mainly caused by exposure to mining asbestos, a natural fibre widely used in building constructions. This untreatable cancer is histologically classified into three subtypes: epithelioid (60% of all mesothelioma cases), sarcomatoid (20%) and biphasic (20%), consisting of both epithelioid and sarcomatoid histology. Current treatment using carboplatin and pemetrexed induces severe side effects and has limited improvement in the survival of mesothelioma patients (3 months). Here, we proposed a new drug delivery system (DDS) based on metal-organic frameworks (MOFs) for mesothelioma therapy. MOFs are crystalline and porous materials formed by the self-assembling of metal ions and organic ligands. Zirconium (Zr)-based MOFs have emerged as promising candidates in biomedical applications owing to their excellent stability and bioavailability. First, NU-901 Zr-MOF was synthesised and a bilayer coating method using phospholipid and biosurfactant was developed to protect the NU-901 from aggregating and degrading in the cellular media. An approved chemotherapeutic drug for mesothelioma, pemetrexed, was then encapsulated into NU-901, followed by the bilayer coating, to achieve a more sustained drug release and improve uptake by A549 lung cancer cells. Live confocal imaging confirmed the colocalisation of bilayer-coated MOFs with 3T patient-derived epithelioid mesothelioma cells and A549 cells with less lysosomal uptake. Next, two more Zr-MOFs with larger porosities, PCN-128 and PCN-222, were synthesised to co-encapsulate the first-line treatments for mesothelioma, carboplatin and pemetrexed. The surface functionalisation process was then simplified from two steps (bilayer coating) to one step using a phosphate anchored polyethylene glycol (PEG) to improve the stability of MOF in three types of cellular media. The number of cell line was also expanded from one to four, including both epithelioid and biphasic subtypes. Compared to the same concentration of pure carboplatin and pemetrexed, dual PCN-222 DDS showed greater toxicity in three mesothelioma cell lines, while PCN-128 DDS was more toxic in one mesothelioma cell line. Furthermore, PCN-128 DDS required shorter time to kill biphasic mesothelioma cells compared to pure drugs. After exploring the role of Zr-MOF in mesothelioma, we realised that introducing toxic solvents and metal ions in the synthesis of Zr-based MOFs was potentially carcinogenic and harmful to the environment. Inspired by the Zr-MOFs syntheses, the PCN-128 linker was functionalised with aldehyde groups and PCN-222 linker with amine groups to develop a new room temperature synthesis protocol of covalent organic frameworks (COF) without adding any metal ions. The COF showed high surface area and crystallinity, but its large size in both dried and suspension states hindered its application in nanoparticle-based drug delivery. Thus, the potential of COF in dye capture for environmental protection was explored. The COF performed as an excellent absorber for dye solution, showing its high affinity to cationic dyes such as rhodamine B and brilliant blue due to its negative surface charge. In summary, Zr-based MOFs have shown great promise in mesothelioma therapy due to their functionalisable surface (bilayer and PEG), dual drug loading (carboplatin and pemetrexed), and improved cytotoxicity to mesothelioma cells. This thesis presented the first milestone in using MOF for mesothelioma therapy, which offers new insights into their applications in animal studies and clinical trials.
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Development of optomechanofluidic resonators
Wharton, David Alan
[Restricted]
Open Access
Dynamics of confined chemical gardens and implications for submarine methane hydrates
(2022) Macedo E Rocha, Luis Alberto
Fascinating patterns may be observed when performing chemical garden experiments, which occur when metal salts come into contact with solutions such as sodium silicate. Hele-Shaw cells, quasi-two-dimensional micro reactors, can be used to reduce the complexity of the system: osmosis is removed when performed with injection, and buoyancy if placed horizontally; the results are thus only dependent on the relationship between flow and chemical reaction. Firstly, we analyse the behaviour of horizontal filaments, one of the main patterns of confined chemical gardens. We model their erratic motion by considering the diffusive supply of ions to the tip, and the spreading of product as the filament advances. We show that these effects lead to an oscillation of the concentration of product at the tip and its internal pressure, causing the filament tip to periodically change direction. We also demonstrate from statistical mechanics that the filament tips grow with a self-organized dispersion mechanism. Effective diffusivities as high as 10−5 m2 s-1 are measured, an efficient transport four orders of magnitude larger than molecular diffusion in a liquid, ensuring widespread contact and exchange between fluids in the chemical garden structure and its surrounding environment. In a second study, experiments were carried out with a vertical Hele-Shaw cell, introducing the effect of buoyancy into the system. The expanded model shows good agreement with the results, while also suggesting that the concentration of the host solution of sodium silicate also plays a role in the growth of the structures despite being in stoichiometric excess. In a third study, novel patterns are described, which grow at flow rates below the threshold for the formation of filaments. We describe and model the evolution of a thin filament wrapping around an expanding “candy floss” structure, forming a new pattern resembling an Archimedean spiral. The effective density of the precipitate as well as the permeability of the membrane were estimated from the results. Finally, in a fourth study, these findings were applied to geological fluid and venting systems of methane. The precipitate filaments grown in the laboratory are used as a theoretical analogue of the spreading of methane hydrates under the seabed. We discuss how this methane venting leads to the formation of marine authigenic carbonate rocks, and for confirmation, we analyse two field samples from the Gulf of Cadiz for composition and mineralogy of the precipitates. We note the implications of this work for hydrate melting and methane escape from the seabed.
Embargo
Zirconium-based metal-organic frameworks as drug delivery systems
Zhuang, Yunhui
Metal-organic frameworks (MOFs) are highly ordered porous materials made up of metal ions or clusters connected by organic linkers. The ease of surface modification for zirconium-based MOFs allows for a variety of designs for targeted therapeutic delivery. This thesis aims to understand and develop the Zr-based MOFs as a drug delivery system. Firstly, a Zr-based MOF, NU-1000, was examined for its ability to encapsulate and deliver a large hydrophobic drug, fulvestrant, in breast cancer cells. NU-1000 was shown to be internalised by the human breast cancer cell line, MCF-7 cells, within 24 hours of incubation. Fulvestrant loaded NU-1000 reduced cancer cell proliferation and inhibited the expression of oestrogen receptors in MCF-7 cells. Although NU-1000 was able to load fulvestrant without defects in its crystalline structure, potential aggregations were observed, which could interfere MOFs’ colloidal stability for drug delivery applications. In order to improve MOFs' colloidal stability, a panel of Zr-based MOFs were surface functionalised with methoxy polyethylene glycol (mPEG-PO3) followed by lyophilisation. These PEGylated nanoMOFs were able to maintain their hydrodynamic diameter in water and PBS compared to their non-PEGylated counterparts. PEGylated nanoMOFs also reduced cytotoxicity in vitro and were taken up by HeLa cells within 24 hours of incubation. Their ability to load, store and delivery drugs were tested using an anti-cancer drug, doxorubicin, where delayed drug-release capability was observed. Since nanoparticles will almost inevitably be in contact with immune cells in the blood circulation system upon administration, human peripheral blood mononuclear cells were used to gain insights into PEGylated MOFs' interactions with the human immune system. PEGylated NU-901 has shown some toxicity towards monocytes but not in T cells, but no toxicity was observed for PEGylated UiO-66, ZIF-8 and PCN-222. The expression of inflammatory cytokines was also examined, where elevated IL-6 was observed for cells treated with PEGylated ZIF-8, a zinc-based MOFs. In summary, Zr-based MOFs have shown many promises to deliver drugs in vitro. Their colloidal stability and biocompatibility can be significantly improved by controlled coordination of mPEG-PO3. This thesis also presented a comprehensive study on MOFs’ immunotoxicity with human primary immune cells, which offers new insights for their potential clinical applications.
Open Access
Improving the efficiency of domestic ovens
Davidson, Jamie
This project aimed to realise and validate a novel design of domestic oven to reduce cooking time and energy consumption compared to a traditional design of domestic oven. The design concept was developed by Dr Mark Williamson of Cambridge Oven Innovation (COI). Initially computational models were used to predict the efficacy of the design concept. Heat transfer coefficients from literature and a computational fluid dynamics (CFD) simulation were imported into a lumped property model, which predicted that the COI design would reduce both energy consumption and cooking time. Three successive physical prototypes were constructed to develop the concept, successively improve the user experience and progress towards a mass manufacturable design. Comparative food trials were undertaken with the third prototype and a domestic fan oven of traditional design. The COI design was able to reduce energy consumption by 30% and cooking time by 60% across a wide range of thermal masses and Biot numbers. The CFD simulation was validated using heat flux and velocity measurements taken in the first prototype. The simulation was found to predict velocity, temperature distribution and convective heat flux reasonably accurately, with a mean absolute error in the convective heat flux of 22%. The radiative heat flux was predicted less accurately, with a mean underprediction of 53.2%. This was attributed to an inaccurate prediction of the temperature of the oven walls, due to: (i) neglecting conduction through interior dividing walls of the oven, (ii) an inaccurate estimate of the overall heat transfer coefficient from the inner wall of the oven to the ambient air and, (iii) reliance on empirical wall functions to calculate heat flux on the walls of the cooking chamber. The CFD simulation was used to investigate the possibility of applying the COI design to a 45 cm high standard kitchen unit, rather than the typical 60 cm height units. The simulation showed that this would increase the heat flux into the food at a given setpoint temperature, and therefore the efficiency and cooking time would be improved. However, the evenness of cooking would be reduced. Potential solutions, namely increasing the number of jet nozzles and changing the size of the jet nozzles, were investigated. This was found to improve evenness at the cost of energy efficiency and cooking time, although they were still less even than the 60 cm design.
Embargo
A Systematic Method for Enhanced Expression of Alcohol Dehydrogenases in Escherichia coli
Mital, Suraj
Expression of recombinant enzymes in bacteria – principally E. coli - is an important method to generate high yields of industrially valuable proteins in short manufacturing timescales. While an extensive understanding of recombinant protein expression at industrial scale in E. coli has been achieved, challenges such as the formation of inclusion bodies, present limitations that inhibit streamlined manufacturing of recombinant enzymes. The lack of consistent workflows to address enzyme solubility issues often results in difficulties in producing high quantities of catalytically active protein. Commercial alcohol dehydrogenases are enzymes often produced in recombinant systems that are primarily employed in industrial chemical catalysis; the formation of inclusion bodies currently limits product yield of these enzymes. The primary aim of the work described in this thesis was to develop a robust strategy for heterologous expression of aggregate-prone industrially relevant alcohol dehydrogenases to improve their solubility in E. coli, thereby alleviating time-consuming troubleshooting steps in the future. A systematic literature analysis was first carried out to identify routinely adopted strategies used to minimise the likelihood of inclusion body formation in E. coli. This revealed that expression methods typically relied upon ad hoc, trial-and-error approaches with limited guarantees of success. This led to the emergence of the need to develop a systematic approach to handle this problem. Select identified strategies were then used to guide a more systematic experimental approach to cytosolic protein expression, which considered the impact of parameters such as plasmid design, strain variation, media formulation, and expression conditions. Protein expression at relatively high medium pH (pH = 9) led to a 4.47-fold and 5.46-fold improvement in soluble yield for two alcohol dehydrogenase models. The experimental design was further developed to improve the cellular density of the high pH system through fitness modifications to the E. coli strains of interest via adaptive laboratory evolution; the final evolved strain provided a further 4.15-fold and 4.87-fold improvement to the soluble yield of the system for the two alcohol dehydrogenase models. The evolved expression strain, E. coli ALE strain ‘D9’, showed key biological differences compared to the parental strain, E. coli BL21 (DE3). While E. coli BL21 (DE3) responded to protein expression at high pH by reducing/modifying its ribosomal profile and translational processes, the evolved strain reduced pathways related to energy intensive metabolic processes to maintain cytoplasmic pH in alkaline growth conditions. This evolved strain contained seven key mutations that conferred changes to cell envelope stress responses, morphology, and transcriptional/translational regulation that contributed to its fitness. The final E. coli expression system developed in this thesis ultimately improved the soluble yield of two model ‘difficult to express’ alcohol dehydrogenases. At the same time, the work exemplified an alternative view to protein expression that stresses the importance of understanding how E. coli biology, enzyme physicochemical properties, and fermentation conditions can affect the solubility of a DtE enzyme; furthermore, this work highlights the importance matching the requirements of an enzyme of interest with the characteristics of the expression host employed.
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Data-driven Dynamic Control Scheme for Antibody Producing CHO Cell Cultures in Fed-batch_Online Appendix_Nishanthi Gangadharan
Gangadharan, Nishanthi
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Machine-assisted synthesis and development in pharmaceutical industry
Jorayev, Perman
Machine-assisted synthesis and development in pharmaceutical industry Perman Jorayev Process development of novel chemical transformations is often a laborious and complex task. This is mostly due to the difficulties in identifying the underlying reaction mechanism(s), selection of chemical (intrinsic) and physical (process) parameters that affect the process objective(s), quantifying the nonlinear interactions between them, and lack of data. Reducing the cost and time for development of robust processes from novel chemical transformations, therefore, requires more efficient solutions to address the individual challenges. In this project, we present several new workflows to tackle some of these challenges. First, we explored use of black-box Bayesian optimisation algorithm TS-EMO for optimisation of complex reaction networks, such as the bio-waste crude sulphate turpentine conversion to functional molecules, with no prior mechanistic information. Using Gaussian processes as surrogate models and sampling from the reaction space based on the highest expected hypervolume improvement, algorithm-guided optimisation of eight continuous variables allowed for identification of the experimental Pareto front to account for the trade-offs between the reaction objectives conversion and yield. Then, expanding to the discrete variable space, we developed a solvent recommendation workflow based on similarity and data fusion techniques, and sustainability guides. Based on two solvent libraries, we demonstrated the use molecular informatics-driven workflow on various chemical transformations, and identified a significant overlap with solvent selection tools developed by AstraZeneca and Syngenta. Next, considering all continuous and discrete variables in the reaction, a holistic modelling of Buchwald-Hartwig amine synthesis using DFT-based parameterisation techniques and a dozen machine learning algorithms led to development of highly predictive reaction models. Through several Explainable AI tools, reaction specific descriptors were identified, and their transferability was validated in the laboratory on a similar reaction. Finally, in order to develop a robust process of a sensitive photoredox amine synthesis reaction, we generated a priori knowledge in the form of solubility predictions and measurements, and quantification of absorbance and photon flux. Using a recently developed NEMO algorithm, we demonstrated simultaneous optimisation of continuous and discrete variables for reaction objectives yield and cost. The workflows developed in this project, as demonstrated on multiple case studies, validated their efficiency and use in process development.
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Fouling and encapsulation of clathrate hydrate solutions
Karela, Anastasia
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Open Access
A Framework for Autonomous Process Design: Towards Data-driven and Knowledge-driven Systems
Khan, Ahmad
The chemical industry is positioning itself for a new era of digital research and development. The complexity of product development, along with the influx of information, entail a need for scalable and intelligent decision-making at different levels of the innovation value chain. This thesis tackles the problem of autonomous process design, by combining data-driven, knowledge-driven, and mathematical programming tools within a unified framework. To realise the intelligent system, the process design problem is first reformu- lated as a reinforcement learning problem, wherein an agent can interact with an environment to build flowsheets, receive reward as feedback, and iteratively improve its designs. Further, a hierarchical agent is developed to first identify desired design tasks, then arrange units to achieve them, thus emulating an expert’s planning strategy. The agent is then combined with an ontological tool, i.e. a computer-readable knowledge base of processes, tasks, phenomena, and operational bottlenecks, along with their attributes and relations. This knowledge is processed using predicate logic to dynamically define states, actions, and objectives on the different design levels. The ontology also enables the combination of phenomena to attain intensified multi-functional unit operations. This work represents a step-change in autonomous design by providing a novel end-to-end design procedure. Further development would enable continual, multi-source knowledge acquisition. This will ultimately lay the foundations for next-generation intelligent systems for scalable process design and intensification.
Open Access
Fundamental Studies of Chemical Looping Combustion with Oxygen Uncoupling (CLOU) of Biomass Char
Kwong, Kien; Kwong, Kien [0000-0001-6495-0185]
Chemical Looping Combustion (CLC) is a technique which utilises lattice oxygen contained in solid particles of an inorganic oxide (the ‘oxygen carrier’) to combust fuels, enabling the collection of products of combustion (CO₂ and H₂O) undiluted with nitrogen. It offers, therefore, an attractive method for capturing CO₂ for use or for sequestration in the Earth. This Dissertation concerns the combustion of biomass chars in a fluidised bed using Chemical Looping Combustion with Oxygen Uncoupling (CLOU), a variant of CLC in which the oxygen carriers can release gaseous oxygen. In CLOU, the gaseous O₂ released from the oxygen carriers reacts heterogeneously with the particles of char, producing a mixture of combustion products, CO and CO₂. The CO, in turn, can react either with the gaseous O₂ or heterogeneously with lattice oxygen in the carrier particles. The overall objective of the research described in this Dissertation has been to investigate how the presence of the CLOU particles affects the rate of combustion, and hence, the burnout time of biomass char, an important fuel for future power generation, but which has received scant attention in the literature. To scrutinise the influence of the CLOU particles, the ratio of CO to CO₂ when combusting biomass char had first to be established experimentally, because that ratio affects fundamentally the transport processes occurring in CLOU combustion, e.g. the external rate of transfer of O₂ to the surface of the char particle. However, information about CO to CO₂ ratios when combusting biomass chars is lacking in the published literature, with previous investigators assuming that ratios were similar to those produced by coal chars. Experimental measurements of the ratio of CO to CO₂ from various types of char were undertaken using a thermogravimetric method. The experiments were conducted at known temperatures between 973 and 1173 K, with a range of partial pressures of O₂ (pO₂) from 0.0057 and 0.023 bar. The CO to CO₂ ratios for chars derived from biomass were significantly different to those for chars derived from coal. Thus, models that utilise the values of CO to CO₂ ratio measured from coal chars will be erroneous if applied to predict the combustion performance of a biomass char. Further investigations substantiated this conclusion insofar as the burnout time predicted in particle-scale models using the ratio of CO to CO₂ of a coal char under-predicted the results from independent burnout experiments with single particles birch char (~ 4 mm dia.) when combusted in a laminar flow of oxidising gas. To evaluate the influence of the CLOU materials on the rate of combustion, particle-scale models have been developed, accounting for the reactions and transport phenomena occurring within, and around, the particle of char in a CLOU arrangement. The char studied here was of a size typically used for fluidised combustion such that external mass transfer could be a significant controlling factor (~ 1 mm dia.). Results from the models were compared with experiments performed by combusting biomass char in a bubbling fluidised bed (i.d. 30 mm) of a CLOU material (a Cu-based oxygen carrier) or inert silica sand. The experiments were undertaken with a partial pressure of oxygen, pO₂, close to the equilibrium pressure of O₂ of the Cu-based oxygen carrier. The results from the model agreed with the experimental observations and revealed that, when a CLOU material is present, there is a significant increase in the combustion rate of char compared to that in silica sand. The increase stems from the release of oxygen by the carrier within the external mass transfer film surrounding the char and was predicted by the theoretical work. The models were also used for investigating the performance of CLOU when the particles of char were combusted under the typical operating conditions used in industrial applications, e.g. where concentrations of CO₂ might be substantially higher, giving contributions from gasification. Finally, the combustion of biomass char in a non-stoichiometric perovskite CLOU material, SrFeO3-δ, was also investigated, both experimentally and theoretically. The performance was compared with combustion in Cu-based CLOU materials and with combustion in an oxygen carrier without CLOU properties, namely Fe₂O₃. Experiments were undertaken at the same operating temperature and pO₂ in the fluidising gas. Significant differences in the burnout time when using the different bed materials were observed and plausible explanations, including accounting for the difference in the oxygen availability of the oxygen carriers, were discussed.
Open Access
Printing protein-engineered paper for biosensors
Gordillo Dagallier, Lorena
The use of paper as a sensing platform has great potential for point-of-care diagnostics in low-resource settings. However, there is a need for approaches that enable a higher level of control and customisation of the paper properties and reduce the cost of production and immobilisation of bioreagents onto paper. This thesis explores whether introducing digital printing into papermaking in combination with the recombinant protein production of paper-binding bioreagents could provide an accessible, scalable tool for the fabrication of affordable paper-based microfluidic biosensors with customised and varying paper properties. A novel printing method based on the localised vacuum-driven filtration of fibre suspensions (‘vacuum-driven printing’) was explored for the direct formation and patterning of engineered paper. An analytical model based on incompressible cake filtration theory was derived to predict the final thickness of the printed paper as a function of printing parameters and showed good agreement with the experimental data. This model enabled the rational design of complex 2D paper patterns with controlled and varying thickness profile that could be formed in a single printing process. The recombinant fusion of a cellulose-binding domain (CBD) into the protein reagent enabled its one-step immobilisation and purification onto paper fibres directly from the crude lysate, significantly reducing the cost and number of downstream processing steps required for the production of the bioreagent. The specific immobilisation provided by the CBD helped retain the protein’s diagnostic activity during air drying and long-term dry storage, even after four months at 20-37°C, when compared to direct physisorption of the protein reagent onto paper. The protein-bound fibres could be directly patterned with the vacuum-driven printing technique into the detection zone of a paper-based assay, forming bioactive paper. A proof-of-concept paper-based microfluidic biosensor was fabricated in a single vacuum-driven printing process, using unmodified paper fibres to form microfluidic channels of varying thickness, and protein-bound fibres to form bioactive paper at the detection zone of the assay. This research demonstrated a simplified, accessible, and lower-cost pathway to the production of paper-based biosensors with customised properties.
Open Access
Colloidal phoretic motion
Rees-Zimmerman, Clare Rebecca
Phoretic motion refers to the transport of particles up or down the concentration gradient of a different solute and can be exploited to control particle motion. This thesis seeks to understand the relative importance of different phoretic effects. It examines phoretic motion in the context of concentration gradients in a drying film, to understand which phenomena govern the stratification of particles in the final dried structure. A colloidal hydrodynamic model valid up to near close-packing, is derived and solved numerically to model diffusion, excluded volume diffusiophoresis, hard sphere and Derjaguin–Landau–Verwey–Overbeek (DLVO) interactions in a drying film. Excluded volume diffusiophoresis is found to act to promote small-on-top stratification, causing fluxes of a similar magnitude to diffusion. The hard sphere interaction model predicted regimes with small- or large- on-top stratification, in addition to a new small-large-small sandwich layering regime. However, its predictions did not quantitively agree with x-ray scattering results of dried silica and latex films. A Hele-Shaw cell experiment measured the flux of latex particles down a concentration gradient of silica particles. The latex motion was arrested by increasing salt concentration, consistent with electrolyte diffusiophoresis driven by silica and its stabilising counterion. This also implies that electrolyte diffusiophoresis is more significant than excluded volume diffusiophoresis for these colloids. The DLVO interaction model showed that by setting the particle surface potentials and salt concentration, we can select either small- or large- on-top or no stratification. Overall, we learn the importance of interactions and electrolyte-driven diffusiophoresis, overlooked in favour of hard sphere interactions and excluded volume diffusiophoresis in the literature, with further work suggested in modelling electrolyte-driven diffusiophoresis in drying films.
Open Access
Terahertz spectroscopy to explore the role of vibrational dynamics in systems with varying structural order and disorder
Kölbel, Johanna
Understanding the processes occurring in materials with varying structural order and disorder, such as pharmaceutical mixtures, has profound implications for drug formulation and delivery. Pharmaceutically active biological molecules such as peptides, proteins, and antibodies need to be formulated and processed into dry powder form that can be reconstituted quickly in order to achieve long-term storage stability. The biomolecules retain their functional properties by embedding them into an amorphous matrix of suitable small organic molecular glass formers. Degradation mechanisms catalysed by water clusters, the influence of the solvation shell in solution upon reconstitution, and crystallisation processes are important aspects to consider in this context, and this thesis investigates these points using terahertz time-domain spectroscopy (THz-TDS). Glycerol is commonly used to protect proteins during cryo-preservation and its interactions with small amounts of water are important to understand. The onset of molecular mobility, as measured by the infrared active dipoles in glycerol-water mixtures, resulted in increased anharmonic effects, obscured the boson peak, and influenced the vibrational density of states. The effect of the relative water content in aqueous mixtures of glycerol at room temperature was also explored. By utilising four different model biopharmaceuticals, the effect of size on the dynamics of one-component lyophilised products was studied with THz-TDS and differential scanning calorimetry. Anharmonic effects were identified in the spectra and linked to protein jamming in high molecular-weight samples, hindering the increase in molecular mobility with temperature. Two-component lyophilisates of varying sucrose to monoclonal antibody ratio were assessed with terahertz spectroscopy and it was shown that protein jamming at a critical temperature must be associated with the macromolecular structure of the protein itself, that it is not dependent on the presence of any excipient, and that it is not dependent on the presence of water molecules. Even if proteins are stored in dry form, they have to be rehydrated before use without losing their functionality due to misfolding or aggregation. Using terahertz spectroscopy and structural techniques, an increased aggregation rate of α-synuclein (aSyn), a protein associated with Parkinson’s disease, in the presence of NaCl compared to CsI was found to be not due to a change in the structural conformations of aSyn, but due to a reduction in both the water mobility and subsequently the protein mobility. The same method was applied to two other proteins, namely β-lactoglobulin and bovine serum albumin, and the interactions with the surrounding salt solution were found to strongly depend on the protein characteristics. Crystallisation dynamics in aqueous solution were studied using THz-TDS in a transmission geometry on the example of magnesium sulfate heptahydrate. A novel method was developed to perform temperature and concentration calibrations of liquid samples at terahertz frequencies, enabling the studies of local concentration of semicrystalline systems.
Open Access
Innovative Deposition of Corrosion Inhibitors by Abrasive Blasting
Krautsieder, Anke
Abrasive blasting of steel using garnet is a commercial surface cleaning procedure, for removing old corrosion scales and paint prior to the application of new coatings. A certain percentage of the garnet and its impurities remain attached, partially covering the steel. Between cleaning and repainting, seawater aerosol droplets can impact the steel and initiate corrosion, which may subsequently lead to coating failure. An innovative approach for the deposition of corrosion inhibitors on steel is proposed, making use of material deposited on the steel during abrasive blasting. This aims to immediately protect the steel from corrosion after cleaning until repainting, to improve the longevity of coatings. The optimum approach to deposit a suitable corrosion inhibitor onto the steel as part of the blasting process was investigated. A selection of anti-corrosive materials were mixed with garnet and tested in abrasive blasting. Material deposition was analysed by electron microscopy and the inhibition efficiencies of the treatment determined in electrochemical corrosion tests. It was found that the impact of garnet particles effected the required cleaning, whilst anti-corrosive materials were successfully deposited on the steel. A correlation was observed between the proportion of the anti-corrosive agent in the blasting mixture and the amount deposited. Direct addition of tannic acid, a solid-state green inhibitor, into the abrasive improved the corrosion resistance of steel by up to 80% for at least 8 h. Commercial encapsulation products can be repurposed for abrasive blasting, showing inhibition efficiencies of up to 65%. Further encapsulation methods were investigated for corrosion inhibitor retention and performance in abrasive blasting. Silica capsules with various shell morphologies and high mechanical stability were prepared. Post-preparation loading of macroporous capsules, with high void volumes, presented a versatile and efficient way of encapsulating inhibitors that cannot be directly mixed with garnet, such as benzotriazole (BTAH). After blasting, BTAH was released from deposited capsules upon contact with a solvent and spread to cover the steel surface, as required for corrosion protection. The extent and strength of adsorption of BTAH from toluene onto steel, iron oxide, garnet, calcium carbonate and silica are presented, including solution self-association corrections for the inhibitor. X-ray photoelectron spectroscopy, sum-frequency generation spectroscopy, and quartz crystal microbalance measurements supported the solution depletion conclusions: for low bulk concentrations, BTAH preferentially adsorbs onto steel, even in the presence of other minerals, due to the increased adsorption strength. With higher availability of BTAH, the other substrates can adsorb significant amounts, in fact, more than S355 steel. However, under this regime, the steel and iron are still expected to have a complete monolayer of BTAH and the corrosion inhibition efficiency should not be affected.

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