Chitin, a homopolymer of ß-(1-4) linked N-acetyl D-glucosamine units, and its deacetylated derivative chitosan have unique properties that may allow their utilisation in a diverse array of high-value applications. Currently chitinous materials are commercially produced from the waste products of the seafood processing industry, this supply is seasonal and the extraction procedures required harsh, resulting in products with heterogeneous characteristics. In this work novel methods of extraction of chitinous material from the dry fungal biomass remaining from the large-scale fermentation of Penicillium chrysogenum in the penicillin manufacturing industry were investigated, with the aim of avoiding or minimising the harsh chemical treatments. This work was carried out in partnership with Angel Biotechnology, who produce penicillin commercially and provided the waste biomass. It was determined that the chitinous material present in this biomass was too intractable for this to be a suitable commercial source of chitin, as large quantities of non-chitinous polysaccharide impurities remained in the product. Attempted enzymatic degradations of the fungal cell wall did not increase the level of purity of the extract. Comparison to other fungal sources of chitinous material indicated that P. chrysogenum does not provide the most efficient source of chitinous material. During the course of these studies it became apparent that there is no agreed literature procedure for the determination of the degree of deacetylation (DDA) of chitinous material, this characteristic is essential in determining the physiochemical properties of the polymer. In reviewing the procedures available we concluded that (^15)N solid-state NMR offered the most reliable method, however, its use was limited by the low natural abundance of (^15)N. We therefore developed a novel, efficient and directed strategy for the (^15)N labelling of chitinous material in fungal cells walls. This allows the direct determination of the DDA of chitinous material in whole fungal cells without the need for lengthy extraction procedures. The whole cell CPMAS ssNMR techniques developed may find many applications, such as monitoring cell wall biogenesis in response to varying nutrient conditions. Additionally, this may allow the rapid screening of fungal species to determine the concentration and DDA of chitinous material.

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The aims of this study were i) To optimize a liquid culture media for <I>D. congolensis, </I>ii) to develop methods of preparing and examining excretory-secretory products (ESP) in culture filtrate, iii) To ascertain the importance of antigens in ESP, released by <I>D. congolensis </I>hyphae <I>in vitro, </I>in immune response to <I>D. congolensis </I>infection. Following initial trials, the basic medium used was a commercial preparation of RPMI 1640 containing Hepes buffer. Several chemical supplements were examined, alone and in combinations for their effect on the growth of two <I>D. congolensis </I>isolates: A Scottish isolate (A5N) from a case of lumpy wool in a sheep and a Ghanian isolate (Gh'89) from a case of bovine dermatophilosis. An optimal synthetic serum free liquid culture medium was obtained by supplementing RPMI 1640 with 0.001 per cent ferrous sulphate, 0.05 per cent sodium pyruvate and 0.025 per cent sodium metabilsulfite (RPMI-FPM). In this medium the growth of the two isolates was similar except that the Scottish isolate rarely completed its life-cycle. Excretory-secretory products (ESP) of the two isolates were prepared from supernatants of cultures in serum-free liquid medium. The peptides in ESP were separated by SDS-PAGE and their relative molecular weights (MWT) calculated. The quantity of protein was greater in 48 hour than 24 hour cultures, the former was used to characterize the ESP. The ESP of A5N contained 15 peptides with MWT from 15-160 kDa. The ESP of Gh'89 contained 15 peptides of approximately (± 2 kDa) the same MWT as those in A5N ESP, a further five peptides in the range 100-200 kDa and two at 94 and 82 kDa. The molecular weight peptides (>90 kDa) of both isolates were susceptible to the action of proteases in the ESP, they were present only in samples containing protease inhibitors. In terms of quantity the dominant peptides in both isolates had MWT 67/70 and 54/56 kDa, the latter was not present in samples prepared by ultrafiltration. Both isolates consistently produced a group of five bands having MWT between 24 and 33 kDa. Doublets of peptides were more common in Gh'89 ESP than A5N ESP, occurring at for example 25/26 kDa and 30/31 kDa. Comparison of ESP peptide profiles with those from sonicated hyphae and filaments showed that the ESP are not products of cell lysis or breakdown of cell walls.

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The objective of this project is to investigate the potential of a novel porous silicone rubber matrix material to immobilize the brewing yeast, <I>Saccharomyces cerevisiae</I>, NCYC 1026, so that the resulting man-made immobilized yeast cell particles, with thin disc geometry, (10mm diameter by 1 mm thick), can be used at high immbolized cell biomass loadings in brewing vessels. A simple engineering modification of a conventional non-mechanically mixed brewing vessel was developed, the Closed Loop Swirl Bioreactor, (CLSB) to induce mixing of the liquid phase in the vessel, so as to suspend the immobilized yeast discs and reduce the liquid phase mass transfer limitations, thereby existing equipment to be used for immobilized yeast brewing. The kinetic state of the immobilized yeast cells was measured independently of the immobilized cell batch bioreactors, using a mathematical model for free call liquid phase batch culture and a set of time course data from a single batch experiment. Mathematical models have been developed for the design of commercial immobilized cell batch bioreactors and the testing of the performance of experimental bioreactors. Due to the complexity of time based batch culture rate processes, and the additional complication of the solid phase mass transfer limited immobilized biomass hold-up, the rate mass balances were integrated numerically. Dimensionless plots of the time based design performance response of the batch bioreactor model were used to determine the key importance of the interacting factors of colonization of, and leakage from, the solid phase matrix to batch bioreactor operation. The immobilizing matrix was found to enhance and stabilize yeast cell kinetics by protecting the cells from changes in the external liquid phase culture environment. The porous silicone rubber is therefore a durable and biocompatible immobilizing matrix with particular reference to brewing. The conclusion and observations made in this research have direct relevance to the industrial application and development of immobilized cell batch bioreactors. Recommendations for future research have been made.