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Palm oil is industrially fractionated on a large scale to yield a liquid olein (OL) product composed primarily of low melting triacylglycerols (TAGs) and a solid stearin (ST) product primarily of high melting TAGs. The physical and chemical properties of these fractionated products differ greatly from the original oil, and have added value. The aim of the work presented in this thesis is to study the partitioning of TAGs during the fractional crystallisation of palm oil and how this relates to their theoretical thermodynamic driving forces for crystallisation. Palm oil was studied under isothermal, non-isothermal and postcrystallisation stepwise remelting conditions. Filtered OL and ST products from the experiments were analysed for their TAG compositions by High Performance Liquid Chromatography (HPLC). Raw composition results showed fully saturated TAGs partitioning significantly to the ST phase, but little difference was observed in the compositions of the more unsaturated TAGs between the OL and ST (it would be expected that these would naturally concentrate in the OL). These observations are attributed to high levels of entrained liquid in the filter retentate, which has also been previously reported in the literature. A correction method based on the assumption that no triunsaturated TAGs should be able to crystallise to any significant extent was proposed to recalculate "true" ST compositions. These calculations indicated very high levels of entrainment (with the retentate possessing more liquid than solid), with typically only about 10% of palm oil TAGs crystallising despite forming a thick slurry. Although this assumption has not been directly verified, the corrected compositions showed behaviour that was very consistent with that which would be expected from thermodynamic driving force considerations. In the isothermal and non-isothermal studies conducted, the corrected ST composition revealed that PPP and other saturated TAGs showed the fastest transformation into the ST phase, followed by POP and other monounsaturated TAGs which predominated only once the saturated TAGs had been depleted from the OL phase. Slightly higher concentrations of PPP were achieved at higher isothermal temperatures (in isothermal studies) and lower cooling rates (in non-isothermal studies). Remelting studies on palm oil revealed that the melting process was largely dominated by trisaturated TAGs. This work has also demonstrated that the Focused Beam Reflectance Measurement (FBRM) technique was capable of detecting particle size and population numbers within the crystallising palm oil system and is a useful probe for detecting multiple events occuring in the crystalliser such as nucleation, melting, agglomeration and deagglomeration.

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The use of chicken manure to enhance the biodegradation of total petroleum hydrocarbons (TPH) in composting bioremediation was investigated to help develop an improved understanding of the chemical, biological and toxicological processes involved. Treatability studies combined with an extensive suite of laboratory analyses were designed and undertaken whereby naturally contaminated oil refinery sludge was either amended with chicken manure or left unamended for a total duration of 90 days. The effects of chicken manure on the biodegradation of fractionated aliphatic and aromatic hydrocarbons, differentiation between biostimulation and bioaugmentation effects of chicken manure, and the potentially detrimental effects of chicken manure on the bioremediation process through the introduction and adverse proliferation of non-hydrocarbon degrading microorganisms and the potential introduction of compounds that may elicit toxic effects on hydrocarbon degrading microorganisms were monitored over the duration of the treatability studies using a combination of chemical, toxicity and microbial laboratory analyses. This study found that the addition of chicken manure enhanced the degradation of C9-C12 aliphatic hydrocarbons. It was found that this reflects a combination of biostimulation and bioaugmentation effects and that volatilisation was minimal. This investigation also found that the addition of chicken manure can have positive effects on bioremediation as evident by the enhancement of conditions for microbial growth and/or activity, introduction and enhanced growth of potential hydrocarbon degrading bacterial populations, and the enhanced reduction in toxicity of methanol extractable hydrocarbons. However, it was found that the addition of chicken manure was seen to cause an increase in toxicity of total leachable compounds, which may present a risk to TPH biodegradation through potential toxic effects on hydrocarbon degrading microorganisms. It is concluded from this study that there is a potential for the use of chicken manure to enhance TPH biodegradation, but that this is likely restricted to low molecular weight hydrocarbons.

The potential of flavocytochrome P450BM3 (CYP102A1) from Bacillus megaterium for industrial chemical transformation and biotechnological application is widely acknowledged. The crystal structures of P450BM3 with fatty acid substrates bound present non-productive modes of binding of substrate with their carbons distant from the iron and the ω-terminal end in a hydrophobic pocket at one side of the active site. Comparison between substrate-free and substrate-bound structures of P450BM3 revealed two pockets (A-arm and B-arm) in the substrate binding channel. In this thesis, A82(I/F/W) mutants in which the ‘B-arm’ pocket is filled by large hydrophobic side chains at position 82 were constructed and characterised. The A82F and A82W mutants have greater affinities for substrates (~ 800-fold) as well as being more effective catalysts of indole hydroxylation than the wild-type enzyme. The crystal structure of the haem domain of the A82F mutant with bound palmitate showed different substrate binding position, in which the substrate is closer to the haem iron than wild-type enzyme. On this basis, a second series of mutants with substitutions at position 438 as well as 82, in which the ‘A-arm’ pocket is modulated by large hydrophobic side chains, were constructed and characterised. The hydroxylation of 11-methyllaurate by wild-type was found to yield traces of the ω-hydroxylated product, which is the first observation of ω-hydroxylase activity of wild-type P450BM3 to date. The mutants with both ‘B-arm’ pocket and ‘A-arm’ pocket filled with larger hydrophobic residues (A82F-T438(V/I/L/F) mutants) demonstrated 2- to 3-fold increases in the formation of ω-hydroxyl-11-methyllaurate. Notably, the A82F-T438L and A82F-T438F mutants also presented a marked enhancement of stereo-selectivity for styrene epoxidation to generate R-styrene oxide (~ 30-fold), suggesting that not only that these mutants of P450BM3 will be valuable catalysts for synthetically useful hydroxylation reactions but also that structure-based rational redesign will be one of the most efficient tools to generate novel biocatalysts.

Open cell micro-architectures are used in a large number of applications, ranging from medical, such as bone scaffolds, to industrial, such as heat transfer structures. Traditionally these structures are manufactured using foaming processes, however advances in additive manufacturing (AM) now allow such structures to be designed computationally and fabricated to a high degree of precision. In this thesis image-based methods are developed for the purpose of generating periodic micro-architectures based on implicit representations. The algorithms developed are shown to be efficient and robust, allowing for the creation of both surface and volume meshes. Methods are presented for the creation of functionally graded structures allowing for arbitrary variations in density between specifiable volume fractions. These algorithms are further extended for domain conforming applications as well as for internal structures in CAD models. By utilising a hybrid approach, imaging techniques can be exploited for the generation of internal structures in CAD models without de-featuring the original external geometry. The structures of interest are also shown to be manufacturable via selective laser melting (SLM). The issue of characterisation, for linear elastic properties, is addressed through the use of a novel homogenisation technique. Large multi-scale problems in irregular domains are divided into smaller sub-volumes using established tetrahedral volume meshing techniques. By performing a series of virtual tests on these macroelements their effective properties can be computed and subsequently used in macro-simulations. The technique is shown to yield results in excellent agreement with the often used kinematic uniform boundary conditions (KUBC). It is also shown how these properties may be used for visualising the distribution in properties over a domain.

Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiation into a variety of mature cell types. Thus, using MSCs for tissue engineering and other medical applications holds many promising advantages over normal somatic cells. However, exactly these characteristics make MSCs more difficult to grow and control in vitro. The aim of this research project was to investigate different culture systems for their utility to expand bone marrow derived MSCs in large quantities. A large scale expansion of MSCs is especially of interest since only a small number of bone marrow derived MSCs are present in donor derived samples, which do not meet the demand for medical applications. In this thesis three different culture systems, static monolayer cultures, stirred suspension cultures, and pour-off cultures, were compared with each other for their ability to support MSCs proliferation while allowing them to keep their full differentiation capacity. Cell samples derived from these cultures were used for cell count, to start a CFU-f assay and to start osteogenic and adipogenic differentiation assays. The highest MSC numbers obtained from the static monolayer cultures were about 460% of the initial cells. The differentiation capacity of these cells was restricted, so they only formed osteoblasts. Furthermore, MSC samples obtained from this culture system were used for proteomic analysis on an electrospray ionisation quadrupol (ESI-qQ-STAR) mass spectrometer with the isobaric tag for relative and absolute quantitation (iTRAQ) method. This analysis revealed a difference in the proteome of MSCs from different passage levels which is involved in a changing proliferation and differentiation behaviour. In stirred suspension cultures, the increase in MSC number varied for the different culture media. The best result was achieved in MyeloCult® medium with Pluronic F- 68, IL-3 and SCF, however, reaching only 140% of the initial cell density, this result was significantly worse than in the control monolayer cultures. The pour-off cultures supported an increase in MSC number, which resulted in 860% of the initial cell number. In addition, MSCs expanded in this culture system were able to differentiate into ostoblasts and adipocytes. Thus, pour-off cultures are the most promising culture system for large scale expansion of MSCs with high differentiation potential.

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