Poly(3-nitratomethyl-3-methyloxetane) (PNMMO) is a nitrate ester pre-polymer which can be cured with an isocyanate and utilised as a binder in solid rocket propellants; CH3 X[-H2C-C-CH2-0-]nY CH2ONO2 where X and Y are the end groups. Electrospray ionisation (ESI) mass spectra of untreated PNMMO show that it contains a series of low-mass cyclic and linear oligomers incorporating up to 22 monomer units. Oligomers of higher mass appear not to be detectable by ESI mass spectrometry. The relative abundances of the cyclic oligomer ions in the spectra are affected by various factors but the governing influence is the size of the alkali metal cation used to promote ionisation. The cyclic and linear oligomers can be separated from each other using column chromatography and the cyclic species can be individually characterised by ESI, size- exclusion chromatography (SEC) and 13C nuclear magnetic resonance (NMR) spectroscopy. Thermal degradation results in the gradual increase in intensity of two main new absorptions around 1729 and 1550 cm'1 in the solution infrared (IR) spectrum of PNMMO. The band at 1729 cm 1 is attributed to the carbonyl group in a formate ester and the formate proton and carbonyl carbon associated with this compound are clearly visible in the ‘H and l3C NMR spectrum at 8.1 and 162 ppm respectively and can be cross-correlated by 2-D NMR. The absorption at 1550 cm 1 is attributed to the asymmetric stretch of a nitro group attached to a tertiary carbon. We propose that this nitro species is formed by the recombination of NO2 following the loss of N02 and subsequent elimination of CH20 from the PNMMO side-chains. The assignment of this nitroalkane was confirmed by ESI and spectroscopic characterisation of a nearly pure sample of the tetrameric nitro species isolated from pyrolysed PNMMO using column chromatography. The thermal degradation pathways and products of polypropylene oxide) (PPO) were found to bear some similarities to those of PNMMO. ESI and matrix-assisted laser desorption ionisation (MALDI) mass spectra of thermally and photolytically degraded PPO show that oxidation occurs predominantly at the secondary carbon as opposed to the tertiary carbon.

Building on work in the field of rural geography, this thesis considers the changes which have taken place to the ways in which woods are managed and how the market for woodfuel has evolved following the considerable growth in demand for firewood and woodchip over the past decade. Through the use of in-depth interviews and field visits to woods and woodfuel businesses across the Midlands, it is demonstrated that the growth of the market has encouraged private woodland owners to carry out more management. This is a significant change from the latter half of the 20th Century when falling timber prices, and the lack of a market for wood products generally, resulted in many woodlands being neglected. In particular, the woodfuel markets have stimulated the removal and restoration of ‘Plantation on Ancient Woodland Sites’ (PAWS) by creating a market for the products of first and second thinning operations. Woodland owners are now able to make small, but significant, profits from their woods which is a marked change from a decade ago. A principal driver of the woodfuel market is the ‘Renewable Heat Incentive’ (RHI), whereby the Government is subsidising the use of woodchip-fed biomass boiler systems as part of the transition towards a greater use of renewable energy sources. Whilst the Forestry Commission’s principal objective remains the production of high quality timber, this thesis demonstrates it is reviewing how it may contribute to the woodfuel market’s growing share of the UK’s energy sector. In the thousands of small, privately-owned woods across England, the majority of which are owned for amenity and wildlife purposes, the thesis explores how the growing demand for firewood and woodchip is encouraging greater management. The many ways in which woodfuel management is complementary to other management objectives like timber production, wildlife and shooting are highlighted. The changes which have taken place to the UK’s forestry sector with the growing use of woodfuel is contextualised by exploring the extent to which they typify a ‘post-productivist’ shift. In many ways, the UK’s forestry sector appears to have entered a post-productivist era, with less emphasis placed on timber production since the 1980s and more policies to promote wildlife. The doctrine has been widely critiqued by examining the changes to agriculture but the forestry sector has been largely neglected, barring the work of Mather et al. (2006) who claim it has undergone a post-productivist shift. This view is contested by exploring how the growth of the woodfuel markets is promoting greater timber harvesting, as well as affecting how public and private woodland owners view their woods as money-making assets. The market from the perspective of the woodfuel merchants is also explored. Their histories and backgrounds are presented, revealing a diverse community of people with different business aims. Following an analysis of the ways in which merchants operate, from the types of machinery used to the methods of delivery, a typology of merchants is proposed. Whilst a ‘professionalisation’ of the sector has taken place over the past decade, with more standardised units of sale and improved customer service, an informal firewood market exists whereby some customers are unwillingly sold unseasoned firewood. With wood burning becoming a more popular activity in the UK following a surge in the use of dedicated wood burners, people are becoming more knowledgeable about the burning properties of different species of wood. However, the growth in demand for wood has led merchants to question whether the UK’s woods and forests can continue supplying the market at the current rate. Many merchants struggle to source timber for woodfuel and this had led them to supplement their stocks by purchasing from European suppliers. A contrasting view, however, is that the UK has a surplus of timber across its many unmanaged woods, as demonstrated by the Forestry Commission’s most recent national survey. The thesis therefore highlights a perception amongst merchants that the country’s timber supplies are at risk from the growing demand for woodfuel.

Limited fossil fuel resources and the environmental impacts of climate change are motivating the development of sustainable processes for the production of fuels and chemicals from renewable resources. The development of alternative energy sources, such as biofuels, will strengthen energy security and reduce dependence on fossil fuels. n-butanol is a biofuel and platform chemical. n-butanol is an advanced fuel with high energy content, compatible with existing infrastructure. Here the ability of microorganisms to use renewable resources for biofuel synthesis is exploited. In this work use of the thermophilic bacterium Geobacillus thermoglucosidasius is explored for the production of n-butanol. Geobacillus is considered a promising industrial process organism due to its high optimum growth temperature and ability to assimilate various substrates including both hexose and pentose sugars. As a relatively novel process organism, first the development of molecular tools was required to enable subsequent engineering of the host metabolism. Here four reporter assays were developed, three of which can be used simultaneously, providing for extensive analysis of qualitative and quantitative gene expression within the cell. A range of promoters and RBS’ were screened. Extension of the Geobacillus vector series, pMTL60000, with new component parts for each module enabled co-transformation of two plasmids into G. thermoglucosidasius. The molecular tools developed were then applied in Geobacillus metabolic engineering work, with the aim of producing the target molecule n-butanol. Initially a CoA dependent n-butanol pathway, based on naturally occurring production by ABE fermentation, was considered. Following introduction of the pathway further metabolic engineering was employed to improve pathway flux, creating a driving force through the pathway and increasing the substrate pool. Production of 0.137 mM (10.166 mg/l) n-butanol demonstrated proof of concept. Next, the use of genes native to Geobacillus were investigated for improved enzyme compatibility. This approach did not generate n-butanol here. Finally a CoA independent pathway utilising the host’s native fatty acid biosynthesis pathway was considered. Using this approach, butyric acid was produced. Butyric acid can subsequently be further converted to n-butanol however this was not demonstrated here. In addition to metabolite pathway introduction, host strain engineering was carried out with the aim of adaptation towards industrially desired properties. Directed evolution resulted in selection of a strain with an increased n-butanol tolerance of 2.5% (v/v). Such modifications resulted in an improved process organism for biotechnological application. This work provides the first reported production of n-butanol in thermophilic and aerobic conditions. Multiple approaches to n-butanol production are evaluated here. Use of heterologous and native genes are considered. Both CoA and ACP dependent pathways were introduced. Each approach presented advantages and drawbacks. A system compatible for use in Geobacillus has demonstrated proof of concept n-butanol production. Further development is required to increase production to industrially feasible quantities.

In recent decades, fully bioresorbable polymer composites with appropriate biocompatibility and mechanical properties have provided an exciting opportunity to replace conventional metal alloy implants. This work explores the development of a novel, one-step in-situ polymerisation (ISP) process for the manufacture of fully bioresorbable phosphate based glass fibre (PGF) reinforced composites with matrix materials of polycaprolactone (PCL), polycaprolactone-polylactic acid copolymer (PLA-PCL) and polylactic acid (PLA). Composites produced via conventional laminate stacking (LS) process were used as the comparison to demonstrate the advantages that ISP can provide in composites quality. In-vitro degradation in phosphate buffered saline (PBS) at 37 °C, flexural property retention and cytocompatibility were investigated for both LS and ISP composites. Additionally, the composites were degraded under representative flexural loading for high cycle fatigue analysis to understand and predict their lifetime in service and their likely mechanisms of failure. Significantly more robust fibre/matrix interface and uniform fibre distribution along the cross section of the composites were achieved via ISP compared to LS. These enhancements resulted in considerably higher initial mechanical properties (~450 MPa and ~24 GPa for flexural strength and modulus, close to the upper range of human cortical bone properties), prolonged mechanical retention, less and slower water uptake and mass loss profiles for the ISP composites. The flexural fatigue life of the ISP composites was at least 10 times longer than the LS composites counterpart within both dry and wet (within PBS at 37 °C) testing environments. Furthermore, positive cytocompatibility was also found for both the LS and ISP PLA/PGF composites. Conclusively, ISP composites exhibited considerably enhanced mechanical retention and drastically improved media resistance, making those fully bioresorbable composites significantly more favourable as materials for bioresorbable bone fracture fixation devices.

Enzymes are promising stimuli for the development of responsive biomaterials for biomedical applications. Enzymes are inherently present in the biological environment thus cleverly designed materials for biomedical applications may require no external stimuli to ellicit the required material response. They have been targeted as stimuli in self assembly of bulk materials owing to the material changes in chemical composition afforded by the enzyme interaction. The first examples of autonomous self-regulated drug delivery systems have been reported via the development of reversible enzyme responsive materials that undergo a material change regulated by the enzymes in their environment. Although enzyme responsive surfaces have been reported there are no examples of reversible enzyme response surfaces. The surface is the first point of contact between the biological environment and a biomedical device/implant. Improving this interaction will improve the integration of these biomaterials in biological systems and it has been proposed that biomimetic surfaces are a promising method for full biomaterial integration in the biological environment. The body strives towards homeostasis and this is frequently achieved by enzymatic activating and deactivation of proteins in the body. This process is repeatable and reversible. Herein we address the absence of reversible and repeatable synthetic enzyme responsive surfaces towards the improvement of biomaterial integration. We aim to develop a truly autonomous system wherein enzymes present in the environment can interact with the modified surface to mediate a reversible material response. This goal was achieved by modifying surfaces with copolymers that contain the recognition sequence for Casein kinase II and Alkaline phosphatase to undergo enzymatic phosphorylation and dephosphorylation. Co and homo polymers of serine and glutamic acid were synthesised in solution and conformation/composition relationship was determined by analysis with NMR, GPC and FTIR. Polymerisation from the surface with NCA-Glu and NCA-Ser was achieved as characterised by FTIR, ToF SIMS, XPS and WCA. Enzymatic mediated phosphorylation (CKII) and dephosphorylation (AP) was monitored by surface analysis (ToF SIMS), by monitoring ATP to ADP conversion and phosphate cleavage from the surface using luminescence and colorimetric assays. Conformational changes mediated by enzymatic interactions with the surface was monitored indirectly using a FRET system incorporated in the surface modification. The modified surfaces were able to support cell culture and osteogenesis. This project has made advances in several fields, 1) The use of NCA-ROP as a method to modify surfaces with copolymers, in particular for a random/ alternating amino acid sequence. 2) The use of NCA-ROP as a method to develop stimuli responsive surfaces, specifically, this is the first report of an enzyme responsive surface prepared from NCA-amino acid derivatives. 3) The use of enzymes as stimuli, specifically, this is the first report of a reversible enzymatic responsive surface. In this system reversible phosphorlyation and dephosphorylation was monitored via changes in fluorescence output indicative of induced conformational changes.

The female-produced sex pheromone for many aphid species comprises a mixture of the iridoids (4aS,7S,7aR)-nepetalactone (I) and (1R,4aS,7S,7aR)-nepetalactol (II). Plant-derived biosynthesis of I and II, in plant species such as Nepeta spp. (Lamiaceae) and Catharanthus spp. (Apocynaceae), respectively, has received considerable attention due to their role as precursors to many pharmacologically active compounds, including the indole alkaloids. To test the hypothesis that aphids biosynthesize I and II de novo similarly to that elucidated for plants, isotopic labelling techniques have been deployed with oviparous pea aphids, Acyrthosiphon pisum (Homoptera: Aphididae). A range of unlabelled and deuterium-labelled putative sex pheromone precursors were synthesised and applied to aphids via one of two methods; dietary incorporation or topical application. For feeding experiments, A. pisum oviparae were artificially fed using diet sachets. Under autumnal conditions, a simple aqueous 200 g/L D-sucrose solution, into which the compounds were dissolved, was found to sufficiently maintain oviparae. Topically-treated aphids were exposed to methanolic solutions of each compound by application of 0.2 μL onto their dorsal surface. Following treatment, the volatile organic compounds (VOCs) released by oviparae were collected by dynamic headspace collection and analysed by gas chromatography coupled to mass spectrometry (GC-MS) to confirm the presence and position of labelling. Using unlabelled derivatives to establish optimal VOC collection parameters, dietary incorporation of all deuterated putative precursors evaluated failed to achieve deuterium incorporation into lactol I and lactone II. Successful deuterium incorporation, as evidenced by the presence of molecular ions at m/z 172 and 170 (corresponding to 2H4-lactol I and 2H4-lactone II, respectively), was demonstrated following the topical application of [C-8,C-10]-deuterated C-8-oxidised geranyl and neryl derivatives. Conversely, deuterium incorporation was not evident using the citronellyl derivatives, highlighting the importance of 2,3-unsaturation during biosynthesis. Through the synthesis and application of [C-4,C-9-2H5]-labelled monoterpenyl derivatives, it was demonstrated that a kinetic isotope effect was not responsible for the lack of deuterium incorporation following application of deuterated monoterpenols and monoterpenals. To address unclarified aspects of lactol I and lactone II biosynthesis in aphids, such as precursor identity and cyclisation mechanism, the synthesis and purification of 8 hydroxymonoterpenol-8-β-D-glucoside and NADP2H probes was attempted but was unsuccessful. These results suggest that aphids follow a route to lactol I and lactone II that is also utilised by plants such as Catharanthus roseus (Apocynaceae). This work is expected to support future research into fully elucidating aphid sex pheromone biosynthesis, including identifying the genes, and therefore enzymes, involved. Future implications in the field of biotechnology-based iridoid synthesis and pest management are anticipated.

Diminishing fossil fuel reserves and the drawbacks of conventional crop-based biofuels has catalysed recent research into the microbial conversion of lignocellulosic biomass into liquid biofuel. Fatty acids represent the most abundant form of reduced carbon chain in nature, and represent the basic building blocks for the creation of a wide-range of advanced biofuels; such as alkanes, fatty alcohols, and fatty acid methyl- and ethyl-esters. It is hoped that the use of a thermophilic platform strain, that is capable of producing fatty acid-derived biofuels at elevated temperatures, will circumvent some of the challenges faced by established mesophilic organisms such as Escherichia coli or Saccharomyces cerevisiae. Here we describe the heterologous expression of an alkane biosynthesis pathway from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 in both E. coli and the thermophilic production organism Geobacillus thermoglucosidasius. Alkane biosynthesis in T. elongatus BP-1 is facilitated by two enzymes: fatty acyl-ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO): both of which were found to demonstrate a level of activity in vivo at mesophilic and thermophilic temperatures (30 - 52°C). Expression of an alkane biosynthesis operon in G. thermoglucosidasius NCIMB 11955 resulted in the production of ~100 mg OD-1 L-1 fatty alcohols, and an inconsistent formation of minute amounts of heptadecane. Improved titres of alkane may be achievable through the identification and elimination of competing pathways, and a better understanding of n-alkane biodegradation in G. thermoglucosidasius. However, we recommend the continued pursuit of fatty alcohol production using G. thermoglucosidasius as a host. Elimination of several fatty acid degradation (fad) genes in G. thermoglucosidasius was undertaken with the hope of showing an ability to manipulate the cellular pool of fatty acyl-ACP substrates available to the alkane biosynthesis pathway. The combined elimination of two long-chain-fatty-acid—CoA ligase genes (fadD1 and fadD2) resulted in increased levels of pentadecanoic- and heptadecanoic acid. The heterologous expression of a fatty acyl-ACP thioesterase (FAT) from Clostridium thermocellum and from the Aminicenantes candidate phylum (OP-8) was also undertaken in an attempt to manipulate levels of cellular FFAs, although we postulate that observation of a differential phenotype requires the development of a strain completely defunct of long-chain-fatty-acid—CoA ligase activity. Fatty acid metabolism in G. thermoglucosidasius represents a complex myriad of multiple genes that are subject to strong homeostasis. Nevertheless, we present evidence that genetic manipulations of G. thermoglucosidasius are sufficient to bring about changes in the fatty acid profile of cells, and encourage the further genetic characterization of fatty acid metabolism in the organism through targeted gene deletions, with the hope of producing an improved platform strain for fatty alcohol and alkane biosynthesis at thermophilic temperatures.

Access to renewable energy and reduction of carbon emissions represent two major issues facing humankind in the twenty first century and beyond. The underlying driving forces behind both are multi-faceted and often intrinsically connected, ranging from environmental concerns over climate change to improving economic security through self-sustaining energy production. Possible solutions to reliance on non-renewable, carbon-emitting fossil fuels have been explored over recent decades, with significant interest placed on biofuels. Due to ease of integration into liquid-based petrochemical fuel infrastructure, these renewable alternatives have been a consistent topic of both industrial and academic interest. Despite offering renewable energy, conventional crop-based biofuel production has faced criticism due to consumption of land, water and other resources associated with agriculture. Acetogens provide a solution to conventional biofuel production due to their utilisation of carbon monoxide and carbon dioxide gas as carbon and energy sources, rather than plant matter. This allows generation of a range of chemical products from a broad range of sources, including industrial waste gases and gasified solid waste. Acetogens offer the double benefit of both renewable energy production, and carbon emission sequestation. This study outlines the development of genetic tools to provide a foundation for using synthetic biology approaches to improve performance of acetogens as industrial chassis. Specifically, development of tools and techniques for the acetogen Clostridium autoethanogenum are described, with further applications of such technology to other Clostridia.

Clostridia are widely recognised as organisms of biotechnological importance. This potential, however, cannot be fully exploited until reliable methods have been developed for the transfer of genetic information into and between members of the genus. As with other Gram-positive bacteria, attempts to develop host:vector systems have focused on protoplast transformation procedures. This approach, however, has been hampered by the lack of suitable plasmid vectors. Studies have been initiated to construct such a vector. As a potential source of a clostridial replicon, the cryptic plasmid of C. butvricum NCIB 7423 (pCBlOl, 6.05 kb) has been examined. The complete nucleotide sequence of pCBlOl was determined and it's minimal replicon characterised together with the elucidation of some of the mechanisms involved in it's replication. The erythromycin resistance determinant (Emr) of the Grampositive R-factor pAM/3l was chosen as a selectable marker and the entire nucleotide sequence of this gene was determined. The Ferredoxin (Fd) gene of Clostridium pasteurianum was cloned and employed in the construction of an expression cartridge. The effectiveness of this cartridge in promoting the expression of heterologous genes was examined using a promoter-less xvlE gene. The conjugative Streptococcus faecalis plasmid pAM/Jl was used to mobilise vectors carrying the xvlE gene (under the transcriptional control of the Fd promoter) into Clostridium acetobutvlicum where the gene was expressed. This represents one of the first reports of directed gene expression in C. acetobutylicum.

This thesis describes various applications of high field n.m.r. for the elucidation of coal structure, and concentrates on discovering the chemical environments of the heteroatoms in coal i.e. those elements other than carbon and hydrogen. Chapter 2 describes how hydroxyl groups may be converted to trimethyl siloxyl groups, observation of which by 29si n.m.r. allows one to determine the types of hydroxyl functionalities present in the original material. Such derivatization methods are well known. However trimethyl siloxyl groups give 29si resonances that have predictable chemical shifts and so the derivative spectra of complex coal tars and extracts are easy to interpret. Using this technique we have investigated the nature of hydroxyl groups in a rank series of coal tars. This has given us some insight into the way the hydroxyls are affected by coalification. Chapter 3 is a demonstration of the use of n.m.r. as a detector for H.P.L.C. This has two principle novelties over previous work. Firstly it does not involve building a special probe and secondly, the data is presented as a contour plot of intensity vs. retention time and chemical shift. This method has been used to investigate the nature of several tar fractions and complements the derivatization technique of chapter 2. The final chapter shows how the nature of sodium in coal was determined by 23Na MAS-NMR. By using the information contained in the chemical shift and linewidths of raw and dried coal it was concluded that sodium exists bound to the surface of coal pores by oxygen functions such as carboxylates and phenoxides. It does not exist as sodium chloride microcrystals as once thought.