There is an increasing need to produce fuels and chemical commodities from renewable resources. Current efforts have been mainly focused on liberating sugars from plant-derived lignocellulosic feedstocks. However, lignocellulosic materials have naturally evolved to resist their microbial and enzymatic degradation and this poses a major problem. Due to these difficulties, alternative feedstocks derived from biomass gasification have recently become a major focus of research. The gasification process generates mixtures of hydrogen, carbon monoxide and carbon dioxide, known as syngas, which can be utilised by some autotrophic bacteria as the sole sources of carbon and energy. Cupriavidus necator strain H16 was chosen as a chassis organism for the current investigation as it can grow to high cell densities on CO2/H2 and, under nutrient limiting conditions, stockpiles huge amounts poly[R-(–)-3-hydroxybutyrate] (PHB). The long term aim of the study was to employ metabolic engineering approaches to re-direct carbon flux so that desirable chemicals are produced instead of PHB. Following the establishment of defined growth media, genetic tools, and DNA delivery methods, the natural resistance of the bacterium to a range of desirable target chemicals was tested and 3-hydroxypropanoic acid (3-HP) identified as a suitable target. However, it was noted that C. necator was able to utilise this compound as the sole source of carbon and energy. Hence, several genes involved in the degradation of 3-HP were identified and inactivated through ORF deletion, resulting in strain CNCA13 unable to grow on this compound. However, this strain was still able to co metabolise 3-HP alongside other carbon sources such as fructose or gluconate, necessitating further investigation, including the introduction of additional gene deletions. Some of these deletions belonged to genes or pathways involved in a reductive route for the assimilation of the compound. The inactivation of one of these candidates over the strain CNCA13 led to prevent the co-assimilation of 3-HP alongside fructose. Following strain development, a heterologous pathway designed to produce 3-HP from actyl-CoA in two enzymatic steps was introduced into the organism. The first committed step in this pathway is the carboxylation of acetyl-CoA to malonyl-CoA, catalysed by the enzyme acetyl-CoA carboxylase (ACC). The second step is the reduction of malonyl-CoA to 3-HP, a conversion catalysed by the bifunctional enzyme malonyl-CoA reductase (MCR) or, in some archaea, by the combination of two monofunctional reductases. Genes encoding ACC subunits and MCRs from different bacteria and archaea were codon-optimised, assembled into functional operons and screened for efficient expression in C. necator H16. All genes were found to be expressed, but production of 3-HP could not be observed, even in strains lacking the ability to produce PHB or to consume 3-HP as the sole source of carbon. Thus, further work is needed to efficiently redirect carbon flux through the generated pathway.

With respect to microbiological food safety, beer is thought to be very safe. This is due to the inability of pathogenic organisms to survive in the harsh environment that beer presents, due to low pH, alcohol content and hop acids. However, there are some organisms which have adapted to brewery conditions and can cause off-flavours, hazes or low ethanol yield. The effects of spoilage and subsequent product recall can result in massive economic losses for brewing companies affected. Silver nano particle coatings for pipes and vessels have been suggested as a means of eliminating or reducing contaminants in the brewery. In this study the sensitivity of several brewery contaminants to silver has been investigated. Pichia membranaefaciens, Brettanomyces anomalous, Candida krusei, Hansenula saturnus, Kloeckera apiculata, Rhodotorula mucilaginosa, Saccharomyces ellipsoids var. diastaticus, Lactobacillus brevis, Pediococcus damnosus, were all tested against a range of silver nitrate concentrations (0-1 mM) in YPD, wort and beer. It was found that sensitivity to silver varied between organisms, but no tolerance exceeded 0.55 mM. It was also found that for the majority of organisms, tolerance to silver decreased under simulated brewery conditions i.e. wort, beer and microaerophillic conditions. In the investigation of potential silver tolerance mechanisms, gene microarrays of Saccharomyces ellipsoids var. diastaticus in wort and beer in the presence and absence of silver found that genes most up-regulated during silver stress were those with transmembrane transporter functions. Silver tolerance testing with gene deletion strains of selected potential silver tolerance genes demonstrated reduced silver tolerance for the deletion strains of the HIS1, COX17 and CUP1 genes. All three of these have known functions in copper tolerance. The data collected in this study would suggest that silver (particularily in nanoparticle form) is an effective means of microbial brewery contamination control especially under brewery conditions. However, further study is needed into the effect of silver antimicrobial surfaces on brewery microbial contaminants, silver concentrations needed in antimicrobial surfaces and silver leaching etc.

The seasonal distribution of keratinophilic fungi bas been studied in the soils surrounding an outdoor swimming pool and in soils of summer pens of hedgehogs. The species isolated were conidial and cleistocarpic Arthroderma uncinatum, Trichohyton terrestre, Chrysosorium keratinophilum, Microsporum cookei and Microsporum gypseum. A correlation between some species and. The numbers of hedgehogs in the pens was seen, due to the influence of keratin provided by the quills and scales of the hedgehogs. No such relationship was seen at the swimming pool, although conidial A. uncinatum was seen to decrease when the swimming pool was open to the public in the summer. However, this appears to be the normal seasonal occurrence since a similar pattern of distribution in soils collected from outside the swimming pool was seen. The effects of the addition of fungicides to soil upon the isolation of keratinophilic fungi have been studied, using the hair-baiting technique. The general pattern showed an initial fall in percentage colonisation of baits followed by a fairly rapid recovery to give greater colonisation of greasy baits compared with controls, and a return to the same or less than the control on degreased wool. This contrasts with the normal observation that the majority of keratinophilic fungi grow better on degreased than greasy wool. A study of the keratinophilic flora of soils of different pH values and from coastal regions has been made as ground work for the experimental section. The new intake of students living in halls of residence at Nottingham University was studied in relation to tinea pedis over an academic year. The initial level in October 1972 was 9% by May 1973 it had increased to 24% but returned to 11% in October 1973. In addition to any seasonal influence, there seemed to be a correlation between the sports played by the students and the incidence of tinea pedis. The showers in halls of residence and the University sports centre were thought to be the main points of cross transfer of the infection. In the experimental section, the effects of pH, temperature, fungicides and sea water upon certain keratinophilic fungi have been examined in vitro. Aspects of the nutrition of C. keratinophllum, A. uncinatum and T. terrestre were studied and the most suitable combinations of carbon and nitrogen sources for optimal growth were determined. A study of the competitive saprophytic ability of several dermatophytes was made using various techniques. By use of the fluorescent antibody technique, it was found that A. uncinatum was a good competitive saprophyte in the presence of keratin and can thus be regarded as a true soil inhabitant. Finally, the mating structures of Arthroderma benhamiae, A. tuberculatum, A. uncinatum, Nannizzia cajetani, N. gypsea and N. incurvata were studied using the scanning electron microscope.

Virulence (parasite- induced host fitness reduction) is thought to correlate positively with pathogen reproduction rates, but its relationship with pathogen transmission is likely to be determined by a trade- off between the costs and benefits of harming hosts. This project aims to investigate factors which affect host-parasite interactions and particularly those which may play a role in virulence evolution. In doing so, it describes experiments carried out using a monogenean ectoparasitic flatworm (Gyrodactylus arcuatus) and its three-spined stickleback (Gasterosteus aculeatus) host. Populations of this fish species experience a range of both environmental and ecological conditions. Such environmental heterogeneity has been found to drive changes in fish phenotypic traits such as morphology, behaviour, life history and physiology which may consequently affect parasite fitness. I demonstrate that under these conditions, parasite strains from different host populations evolve variation in virulence levels. I also reveal that coevolution in this host parasite system is likely to lead to local adaptation of G. arcuatus at the host population level. I show that competition between parasite species sharing a single host leads to increased parasite reproduction rates, but it shortens the infection time which may be due to earlier stimulation of host immune responses. I show that virulence is neither influenced by the population density, immunity (epidermal mucus), social behaviour of fish hosts nor the natural parasite life expectancy. Lastly, I find that virulence in this system is negatively influenced by the density of stickleback predators and positively associated with loch water pH. Taken together, these results suggest that in this host parasite system, both ecological and environmental factors which drive phenotypic changes in fish hosts may evolutionarily feedback to affect parasite virulence.

The removal of yeast biomass (cropping) at the end of fermentation to inoculate a subsequent fermentation (serial-repitching) is common practice in the brewing industry. Between successive fermentations cropped yeast is stored as a slurry in cooled storage vessels under anaerobic conditions until required for subsequent use. Maintenance of yeast quality during storage is critical for subsequent fermentation performance. An assumption is made in brewing that all strains benefit from storage at 3-4°C. To test this assumption a model working system was initially established to assess cooling times of lager yeast in different suspension media. Preliminary investigations focussing on freshly propagated yeast slurry demonstrated that whilst the deleterious effects of extremely high storage temperatures on lager brewing yeast physiology was in line with expectation, utilization of traditionally recommended storage temperatures does not necessarily benefit yeast physiology when compared to slurry maintenance at slightly higher temperatures. Genome-scale transcriptional analysis in slurries cropped following an initial fermentation suggested that lager yeast might experience cold stress during slurry maintenance at typically recommended storage temperatures. In contrast, maintenance of lager yeast at a slightly higher storage temperature, in this case 10°C, yielded no adverse impact on key indicators of brewing yeast physiological state or on subsequent fermentation profiles following repitching into fermentations. Whilst these observations were not made using full production scale, they do indicate that optimal storage may not be currently being deployed for brewing yeast at full scale.

The growth of micro-organisms in foods is different from that in axenic liquid culture in laboratory media. In natural environments, including food, micro-organisms generally grow in mixed culture and in close proximity to each other, because of which antagonistic or synergistic interactions can occur. To elucidate the behaviour of bacteria within food matrices an understanding of the food structure is required, as foods are complex ecosystems on the micrometer scale. Most processed foods are emulsions and as such are highly structured heterogeneous environments. Antibody-linked probes can be used for the immuno-location of micro-organisms or their products within food matrices to demonstrate the sites at which growth occurs and elucidate the possible bacterial interactions with food components. The aim of the project was to raise antibodies to spoilage Pseudomonas species and to use the developed antibody-linked probes to follow psychrotrophic spoilage Pseudomonas within heterogeneous foods. By using antibody-linked probes the natural spoilage of milk and milk products can be followed along traditional lines examining extrinsic parameters but with the additional benefit that the major spoilage organisms can be located within the mixed natural flora. The use of antibodies in this way facilitated the study of a defined natural population and surmounted any adaptive problems associated with introduced organisms. An oil-in-water near-foodgrade model was developed to investigate the growth of Pseudomonas as it overcame some of the technical problems of using natural cream. Pseudomonas species, which grew as colonies within the near-food-grade model, were visualised using fluorescently-labeled antibody-linked probes. Pseudomonas used to raise the antisera were isolated from psychrotrophically spoiled food and characterised together with isolates retrieved from the environment. The phenotypic characterisation of Pseudomonas using classical biochemical tests and API 20NE test strips (BioMerieux) did not produce definitive identifications of the unknown isolates. Nutritional screening of the Pseudomonas isolates using commercially produced standardised test microtitration plates (Biolog MicroPlate TM), that contained 95 carbon sources, was carried out. The data produced from the test microtitration plates were analysed using numerical taxonomic methods. The relatedness of the Pseudomonas isolates was strongly influenced by the source from which the test isolates originated and did not definitively identify all of the unknown isolates tested. Molecular techniques, ribotyping and amplified ribosomal DNA restriction analysis (ARDRA), based on the genomic fingerprinting of the 16S rRNA gene were evaluated to aid the definitive identification of the Pseudomonas isolates but needed a more extensive data base to be useful. The difficulties encountered in phenotypically identifying food and environmentally isolated Pseudomonas species stems from the fact that the Pseudomonas genus is now classified according to its ribosomal DNA homology. The classification of the species within the Pseudomonas genus is still under review. Robust phenotypic criteria for the identification of all the species within the genus have not to date been defined. In this study, the association of phenotype with environmental source of isolation (whether characterised by nutritional studies or by antibody cross-reaction) demonstrates clearly that more appropriate phenotypic characterisation is required to allow identification schemes to reflect the underlying phylogeny of this group.