Recombinant protein production (RPP) is a cornerstone of bioprocessing. This study presents novel analytical techniques and production protocols for RPP in E. coli, particularly regarding industrial applications. Flow cytometry (FCM) was used to monitor cell physiology and RPP during production of a fluorescent model protein, CheY::GFP. Further applications of FCM for monitoring RPP were developed: The amyloidophilic dye Congo red was used to identify inclusion bodies produced under high-stress conditions. FCM analysis of transformants on agar plates identified 3 populations of varying fluorescence intensity and the progressive transfer of cells from the high fluorescence population to one of intermediate fluorescence and low culturability. Congo red staining showed this was due to amyloid-inclusion body formation. RPP conditions that minimise physiological stress by reducing temperature and inducer concentration can increase product yields, solubility and biomass yields. The original fermentation protocol used for stress-minimised RPP proved unsuitable for industrial use. Application of stress-minimisation to an industrially-derived protocol using early or late-phase induction and glucose or glycerol as carbon source generated high biomass, total CheY::GFP and soluble CheY::GFP yields. These protocols improved biomass generation, product formation and reproducibility over the original stress-minimised and unmodified industrially-derived protocols and therefore stress-minimisation is of potential industrial use.

Polyethylene terephthalate (PET) films can be advantageously utilised to replace both glass and metal in photovoltaic (PV) devices. However, there remain aspects of their performance in outdoor applications which may be improved upon to meet PV device requirements more efficiently. DuPont Teijin Films (DTF) employ artificial weathering techniques to investigate PET film degradation processes, such as UV degradation and hydrolysis, which occur during the outdoor application of PET films. In this Thesis, a thorough investigation into the modification of PET film properties with exposure to various artificial weathering techniques has been conducted. Techniques including infra-red spectroscopy, gel permeation chromatography, atomic force microscopy and nano-indentation have been employed to improve the understanding of the effects of artificial weathering on PET films. The effects of exposure to high intensity simulated solar radiation have also been investigated and compared with those of the combined degradation agents present during ISO standard accelerated environmental weathering. Surface modifications have been compared with those of the bulk, in particular, surface roughening and microcracking have been investigated in much greater detail than previously in the literature. Finally, the stabilizing effects of including an organic ultraviolet absorber on weathered PET film bulk and surface properties have also been assessed.

Industry operations require a clean plant to make safe, quality products consistently. As well as product quality, the environmental impact of processes has become increasingly important to industry and consumers. Cleaning In Place (CIP) is the ubiquitous method used to ensure plant cleanliness and hygiene. It is therefore vital the system is optimal and efficient. I.e. the correct cleaning agent is delivered to the fouled surface at the right time, temperature, flow rate and concentration. This cannot be assured without effective online measurement technologies. Fryer and Asteriadou (2009) describe how the nature of a fouling deposit can be related to the cost of cleaning. The evolution of three key deposit types has also enabled current fouling and cleaning literature to be easily classified. In the brewery there are many types of soil that need to be cleaned of which the cost of cleaning was unknown. The cost of fermenter CIP in one brewery was found to be £106 k per year. Effective fouling methods for yeast and caramel; and the relationship between flow, temperature, and caustic concentration in the removal of yeast and caramel soils seen in industry has been done. This work has helped determine effective cleaning methods for these soils from stainless steel coupons and pipes. Fermentation vessels have been found by Goode et al., (2010) to have two types of soil: A – fouling above the beer resulting from the act of fermentation, and B – fouling below the beer resulting from emptying the fermenter. The type B fouling below the beer was found to be a type 1 soil that could be removed with water. An increase in flow velocity and Reynolds number decreased cleaning time. An increase in temperature did not decrease cleaning time significantly at higher flow velocities, 0.5 m s-1. Fouling above the beer occurs when material is transported to and stick on to the wall during fermentation foaming. This happens initially and as a result the fouling has a long aging time. This yeast film represents a type 2 deposit, removed in part by water and in part by chemical. Most of the deposit could be removed by rinsing with warm water. At 50°C the greatest amount of deposit was removed in the shortest time. A visually clean surface could be achieved at all temperatures, 20, 30, 50 and 70°C, using both 2 and 0.2 wt % Advantis 210 (1 and 0.1 wt % NaOH respectively). A visually clean surface was achieved quicker at higher detergent temperatures rather than rinsing at higher flow velocity or concentration. This finding suggests most deposit can be removed with warm water and cleaned with lower detergent concentrations. Currently in the brewery 2 % NaOH is used at 70°C. Caramel represents a type 3 soil. When heated it sticks to stainless steel and requires chemical action for removal. Confectionary caramel was cooked onto pipes and coupons and the effect of flow velocity, temperature and concentration on removal determined. At high flow velocity most of the deposit could be removed from the pipe using water. There was no significant difference in the mass of caramel removed by the water however. A visually clean surface was achieved by rinsing at 80°C with 2.5% Advantis. A visually clean surface could not be achieved at lower temperatures at higher concentration, 5% Advantis, or at higher flow velocity. The measurement of online conductivity and flow rate values was invaluable during each experiment. Turbidity values did indicate the removal of yeast and caramel from pipes however offline measurements were required to confirm removal. Caramel removal could be wholly quantified by mass when cleaning pipes. The integration of the turbidity values measured during each rinse correlated well with the mass of deposit removed in most cases. Coupon cleaning was wholly quantified by area . A cost saving of £69 k can be made by optimising fermenter CIP to warm pre-rinsing followed by ambient caustic circulation. An £8 k saving can be made by optimising yeast tank CIP to pre-rinsing only and acid sanitisation. Industry must ensure effective online CIP measurements are made throughout cleaning to describe the process effectively and enable optimisation. It is crucial to have cleaning measurement information to hand because that is how we ensure our customers they are buying a quality product. Also you cannot optimise what you do not measure effectively.

In modern times we have become increasingly reliant on mammalian cell culture for the production of biopharmaceuticals; therefore research aimed at improving the characteristics of the cell-lines being used for recombinant protein production is essential. In this study I have examined the hypothesis that the creation of a CHO cell-line in which the expression of p21 \(^C\)\(^I\)\(^P\)\(^1\)and Bcl-2 could be combined would un-couple cell growth from cellular proliferation resulting in a significant increase in both the rate of production and culture viability. Analysis of key metabolites together with changes in cell volume, total protein and mitochondrial activity indicate that following the initiation of p21\(^C\)\(^I\)\(^P\)\(^1\)-expression cells undergo an increase in their protein synthesis machinery and that the energy, previously required for cell division may be diverted towards cell growth and product formation. In addition to the requirement of cell-lines with high production capacities, the biopharmaceutical industry is under constant pressure to develop growth media able to facilitate high yields without the need for the addition of protein or serum. This means it is often necessary to adapt high producing cell-lines to growth in such a defined chemical environment, a process which has proven to be both extremely long and costly. In this thesis I have successfully developed a method for the swift adaptation of commercially important cell-lines to growth within a chemically defined bio-processing environment. I have shown that the expression of p21\(^C\)\(^I\)\(^P\)\(^1\)is able to reduce the need for extracellular growth factors and that by combining the expression of p21\(^C\)\(^I\)\(^P\)\(^1\) and Bcl-2 it is possible to further reduce the time required for successful adaptation, supporting the well established theory that Bcl-2 plays an important role in apoptotic signaling pathways.

The hydrogen permeability of surface modified Pd\(_6\)\(_0\) Cu\(_4\)\(_0\) wt% (Pd\(_4\)\(_7\)\(.\)\(_3\)Cu\(_5\)\(_2\)\(_.\)\(_7\)at%) membranes have been determined for the first time. Surface modification was accomplished through the deposition of Pd thin films of three different thicknesses (95.5 ± 0.1, 797.4 ± 0.2 and 1,409.6 ± 0.2 nm) on to one side of a range of as-received Pd\(_6\)\(_0\) Cu\(_4\)\(_0\) wt% membrane coated with a 1,409.6 ± 0.2 nm thick Pd thin film positioned on the feed side (445 kPa of hydrogen pressure) and cycled between 50 and 450 °C achieved the highest hydrogen permeability of 1.09 x 10\(^-\)\(^8\) mol m\(^-\)\(^1\) s\(^-\)\(^1\) Pa\(^-\)\(^0\)\(^.\)\(^5\) at 450 °C during the third cycle. This is a 58% increase on the value measured for the as-received Pd\(_6\)\(_0\) Cu\(_4\)\(_0\) wt% under the same conditions. This improvement can be attributed to a Pd-rich Pd-Cu face centred cubic (FCC) phase forming through interdiffusion between the Pd thin film and bulk Pd-Cu membrane as a result of the test conditions used during hydrogen permeability measurements. This introduces a larger hydrogen concentration gradient across the membrane due to the relatively high hydrogen solubility of the Pd-rich Pd-Cu FCC phase resulting in the observed increase in permeability.

A problem with the Polymer Electrolyte Fuel Cell (PEFC) is the expensive platinum (Pt) electrocatalyst. This thesis aims to investigate alloying of Pt with cheaper metals that not only reduce the overall cost but also alter the electronic properties to improve reaction kinetics. A Genetic Algorithm (GA) coupled with Density Functional Theory (DFT) approach has been used to perform structural searches on small Pt clusters doped with early transition metals (M). It is found that varying spin can have significant effects on the minimum energy structures of pure Pt clusters, while doping with early transition metals leads to spin quenching. DFT studies have been performed to predict potential Pt-based alloy nanoparticles that will result in weaker Pt–O interactions. This is achieved by investigating nanoalloys that lead to filling of the Pt d-band. Early transition metals are found to be promising, where donation of electron density from M to Pt results in additional filling of the Pt d-band. The surfaces of pure Pt clusters are found to distort, facilitating fast oxygen dissociation. It is found that the strong Pt-M interactions, which lead to filling of the d-band, can lead to Pt clusters becoming more structurally rigid, which inhibits oxygen dissociation. A search has been performed to find the best compromise for a system that retains flexibility of the Pt surface, to allow fast dissociation while also allowing M to Pt electron donation, leading to filling of the Pt d-band.

There is increasing clinical need for bone substitutes because of the limited supply of autogenous tissue, and the significance of inherited or other bone diseases. The ultimate aim of this study was to form calcium phosphate (CaP) based matrices as bone grafts for medical applications. Amongst CaP based materials, CaP gels made by the sol-gel process have attracted much interest since they can be processed at room temperature allowing the incorporation of environmentally sensitive molecules such as growth factors. CaP gels can be engineered by changing process conditions. There is little previous work however on the effect of drying regimes on the CaP materials formed using the sol-gel process. The objectives of this research were to investigate the influence of drying conditions on the physicochemical properties of CaP gels and the effect of the resultant structures of CaP gels on the function of the incorporated therapeutic molecules. In addition, surface modification of the CaP gels was investigated as a means to enhance biological interaction and also a potential way of creating primary bonds between apatite crystals enabling mechanical reinforcement of the material, which is currently too weak to bear load. This work has confirmed that different drying regimes have a significant influence on the formation of the gel pore structure, with the storage of gel in humid conditions, enabling reprecipitation of an apatitic phase. This variation in pore structure has a significant influence on the catalytic of encapsulated enzymes. In addition, the pH fluctuation of CaP based matrices during processing determines the activity of biomolecules after incorporation. It has also been shown that it is possible to form thiol functional groups on the surface of CaP gels, which could be used in future for mechanical reinforcement or for the attachment of biological moieties.

Hydrogen is seen as a potential fuel for the future; its choice is driven by the increasing awareness of the necessity for clean fuel. Together with the simultaneous development of “green technologies” and sustainable development, a current goal is to convert waste to energy or to create energy from a renewable resource. Biological processing [of renewables] or bioremediation of waste to create hydrogen as a product fulfils this goal and, as such, is widely researched. In this work, an already established process, using a hydrogenase up‐regulated strain ‐ was characterised and the important process parameters were established. This bacterial strain has the potential for industrial‐scale hydrogen production from, for example, waste sugars. Previous work, repeated here, showed that hydrogen could be generated by E. coli HD701 using a two‐phase process (growth in shake flasks, followed by hydrogen production within a bioreactor). Ideally a commercial process would need to be in a single vessel (bioreactor), which therefore resulted in this investigation of the scale‐up of twophase fermentations to 5 L stirred tank bioreactors. Within the initial two‐phase process, shake flask growth in 2 L shake flasks (employing a 50% working volume) achieved a dry cell weight of 1.33 +- 0.1 mg mL‐1 which then, when transferred to a 5 L bioreactor (containing 2 L of culture and 2 L of hydrogen production substrate), achieved a maximum hydrogen production rate of (200 mL h‐1) 150 mL g(dcw)‐1 h‐1. The first step in scale‐up was to simply transfer the process to a bioreactor and see the effect it had on hydrogen production. This approach did not yield any hydrogen and therefore consequent experimentation sought to see if the hydrogen production was growth phase dependant. However all phases of growth evolved no hydrogen upon the addition of substrate. The next approach was to take the conclusion drawn from a literature survey that showed a need for microaerobiosis or anaerobiosis during growth (for mixed acid fermentation to occur) along with a high formate concentration necessary for the transcription of the FHL complex (the hydrogen gas evolving enzyme). For this reason the KLa from the initial shake flask growth (calculated from literature correlations) was applied to the bioreactor. Experiments used to simulate the shake flask mass transfer coefficient (kLa) in a bioreactor did not generate hydrogen; the physical system within the shake flask used for growth in the initial process allows for this to occur, but the consequent process change to a bioreactor did not. This inability to produce hydrogen was concluded to be due to the lack of microaerobiosis/anaerobiosis required for mixed acid fermentation (the metabolic precursor to hydrogen production). The criterion of KLa was inappropriate for scale up in this case due to the physical differences between the shake flask and the bioreactor, as the oxygen transfer within the shake flask is not limited to transfer between the liquid and gas phase (the effect of transfer across the shake flask closure must be considered). This fact led to the novel use of gas blending for dissolved oxygen tension control. Gas blending was used in a bioreactor to track the changes observed during growth in the shake flask. This created a process that mirrored the shake flask in both growth and hydrogen production. The outcome was a dry cell weight of 1.34 +- 0.02 mg mL‐1 and a maximum hydrogen production rate of 200 mL h‐1 i.e. 150 mL g(dcw)‐1 h‐1, exhibiting almost identical process results to the two‐stage process. This characterisation reinforced the necessity for microaerobiosis during growth to allow subsequent post‐growth hydrogen production. Microaerobiosis in the latter stages of growth allows mixed acid fermentation to occur, which was found to be essential for hydrogen production. Process intensification took place by increasing cell density. This was achieved by increasing the medium concentration, then by changing the medium (two differing fed batch media were chosen; each medium used was experimentally linked with multiple feeds) and finally by utilising the novel technique of combining gas blending with fed batch cultivation to ensure microaerobiosis during growth. This, along with the use of a low (\(\mu\)=0.05 h‐1) growth rate for feed calculation, led to an eight‐fold increase in cell density. The low growth rate was employed to reduce inhibitory acetate formation while the multiple feeds were used to investigate nitrate depletion. The maximum increase in cell density led to a hydrogen evolution rate of 1800 mL h‐1, thus producing hydrogen that could be converted into energy at a rate eleven‐fold greater than the rate at which it consumed energy for agitation.

Despite the fact that around 20000 cups of coffee per second are produced worldwide (making coffee the second most-traded commodity in the world), coffee extraction is not well understood yet. This Engineering Doctorate Thesis seeks to advance the fundamental engineering understanding of coffee extraction. This aim is based on the current need of industry to optimise soluble coffee process (as stress on water and energy is increasing), and the growing popularity of On-Demand coffee systems. The macrostructure, microstructure and extraction parameters of roast and ground coffee were investigated. The findings from this study were used in a multi-scale extraction model that portrays the extraction of coffee soluble solids as the combination of phenomena taking place at the particle scale (~μm), and the packed bed scale (~cm). Effective diffusion coefficients in the range of 10\(^-\)\(^1\)\(^1\) m\(^2\) s\(^-\)\(^1\) were shown to offer the better fit to experimental data if a single effective diffusion coefficient is to be used. The model was shown to predict literature extraction data for caffeine, chlorogenic acids and trigonelline in espresso coffees. A new methodology to estimate the permeability of roast and ground coffee in steady state was also developed. Permeability values resulted to lie between 10\(^-\)\(^1\)\(^3\)-10\(^-\)\(^1\)\(^4\) m\(^2\).

The development, characterisation and testing of a novel all-optical, multi-modal preclinical biomedical imaging system is presented. The system aims to provide a new way of accurately visualising the spatial distribution and activity of molecular structures and processes in small animals by combining 3D bioluminescence tomography (BLT; reconstruction-based 3D imaging of internal bioluminescent reporter distributions), diffuse optical tomography (DOT; reconstruction-based imaging of optical parameter distributions) and optical surface capture techniques. The key principle of the imaging system is to use surface capture results to enhance the accuracy of DOT image reconstruction, and to use the results of both surface capture and DOT to enhance the accuracy of BLT. Presented experiments show that the developed system can reconstruct luminescent source distributions and optical parameters accurately and that small animal imaging is feasible with the system.