Carbon monoxide (CO)-utilising acetogens were enriched and the isolate KIST612 was selected for its abilities to tolerate high CO and acetate concentration. The isolate KIST612 was identified as <I>Eubacterium limosum</I> based on the morphological and biochemical characteristics. <I>E. limosum</I> KIST612 produced acetate and butyrate from CO. The optimum temperature and pH for the growth and organic acids formations were 37°C and 7.0, respectively. This bacterium was cultivated on phosphate-buffered basal medium (PBBM) with CO as the sole energy and carbon source. In a batch fermentation using a serum vial, <I>E. limosum</I> KIST612 grew at the initial growth rate of 0.15-0.16 h<SUP>-1</SUP> with <I>K</I><SUB>s</SUB> for dissolved CO of 0.14 mM. When sufficient CO was supplied using a bubble column reactor, the maximum growth rate of <I>E. limosum</I> KIST612 was 0.23 h<SUP>-1</SUP>. The bacterial growth rate was reduced in the presence of acetate. A membrane reactor was employed to allow cell recycling continuous sparging CO fermentation to organic acid product removal. The reactor system used was a bubble column type reactor, and the overall volumetric CO mass transfer coefficient (<I>k</I><SUB>L</SUB><I>a</I>) of the reactor was 72 h<SUP>-1</SUP>. When the dry cell weight was 5.25 g/L in the reactor, the bacterial cell concentration did not increase at a CO partial pressure lower than 74 kPa, though CO was consumed with organic acid produced. At this stage, supply of CO mass transfer rate was lower than CO requirement to support maximum cell growth, but higher than that to maintain culture. Since CO was supplied higher than maintenance requirement under atmospheric CO pressure, bacterial cell concentration increased to 9.5 g/L.

Using <I>Aspergillus awamori </I>ATCC 14335 in flask cultures, the growth medium was optimised for the production of extracellular glutaminase. The medium consisted of simple salts with glucose and soybean flour. Factors controlling the expression of glutaminase were the glucose : soybean flour ratio; the lower the ratio, the higher the levels of glutaminase. High levels of ammonia completely inhibited glutaminase production. The levels of magnesium sulphate and phosphate also influence production. Using these methods, initial activities of less than 200 nMoles/min/L were increased to 2000 nMoles/min/L in optimal conditions. The glutaminase was found to be primarily an intracellular enzyme and extracellular activity is a leakage product of the cell. Studies of disruption revealed ratios of intracellular to extracellular activities of 20:1. During growth in large scale cultures, this ratio was found to vary from under 5:1 to over 20:1. Using 5 and 50 litre bioreactors the scale-up and production of glutaminase was investigated. Studies of the time course of the fermentations for glutaminase production supported the control mechanisms inferred in the shake flask studies. Intracellular glutaminase production peaked in a narrow window between glucose depletion and high concentrations of ammonia in the growth medium. Extracellular activities were detected after the intracellular production had peaked. Glutaminase levels varied by at least a factor of two between sub-optimal and optimal harvesting conditions (i.e. intracellular activities of 7000-18000 nMoles/min/L). Intensification of the fermentation was limited by the levels of biomass which could be grown in the bioreactor. This was mainly due to the poor mixing associated with high concentrations of mycelia. Using a crude enzyme preparation, the enzyme was characterised and compared with enzymes in the literature. This showed that the enzyme was substantially more stable than those previously published e.g. half life (141 minutes at 40°C for the intracellular enzyme), Km values for the intracellular/extracellular enzymes were 0.432 nM and 0.330 mM respectively.

<i>Bacillus macerans</i> is capable of producing, under certain conditions, acetone and ethanol by fermenting a wide variety of carbohydrates. Little work has been done to investigate the system since the first two decades of this century and the aim of this study was to assess the potential use of <i>B.macerans</i> for the production of solvents together with an investigation of the factors controlling solventogenesis. Typically solvents were produced after an initial acidogenic growth phase in which the acetic was formed with a concomitant drop in pH. After an initial examination of the cultural characteristcs of the organism, the study involved the investigation of the conditions of solvent production in 3 types of pH controlled fermentation system, i.e. batch, single and two-stage continuous cultures. The main theme of these investigations was to determine the influence of glucose concentration and pH on the production of solvents. The results show that good concentrations of solvents, up to 250-300 mM ethanol and 80-100 mM acetone from 5% w/v glucose, could be produced in both batch and two stage continuous systems. However the single stage continuous culture gave poor results with low carbon recoveries and lower solvent concentrations (ethanol-acetone). This was thought to be largely due to the selective pressures associated with such a system and the incompatabilities of such a system with non-growth related (secondary) end product formation. Carbon recoveries for all systems were low especially in single stage continuous cultures, where lactic acid was an additional product. It was also suspected that additional undetected non volite products are produced. The volumetric productivities of three solvent producing systems at pH 6 on 5&37 glucose were 0.253 g/h/l for ethanol and 0.083 for acetone in batch; 0.699 g/h/l for ethanol and 0.088 g/h/l for acetone in continous; 0.702 g/h/l for ethanol and 0.229 g/h/l for acetone in a two stage system.

The objective of this project was to investigate the effects of the relative supplies of oxygen and glucose on the kinetic activity of the yeast <I>Saccharomyces cerevisiae</I> NCYC 1026. This study was initiated from observing yeast cells immobilised in the silicone rubber matrix material, <I>immobaSil</I> which has the property of high solubility of oxygen, reported to be eight times that of oxygen in water. Hence, would exposure of the yeast, as free cells, during storage to relatively higher or lower supplies of oxygen to glucose, lead to detrimental and irreversible changes in the kinetic activity of the yeast cells? By identifying the effects of storage conditions on yeast kinetic activity, suggestions could made as to how best to store yeast cell lines so as to promote desired levels of kinetic activity, i.e. to engineer the yeast cells to respond manner in industrial bioreaction applications, and to be able to predict the performance of immobilized cells. The yeast cells were stored as several different lines (Master Lines) which were exposed to varying supplies of oxygen and glucose during storage. Using aseptic handling techniques, these Master Lines were kept successfully for up to 1000 days continuous storage. From time to time, samples of yeast from these Master Lines were taken to produce an inoculum for free cell aerobic batch culture experiments, (Batch Runs), carried in a Stirred Tank Fermenter (STF). These batch culture experiments were conducted mainly under the initial conditions of 1000 mg/l glucose in a chemically defined liquid medium, an aeration rate of 41/min, (1 vvm), a temperature of 25<SUP>o</SUP>C and pH 4.5. The variations in the relative supplies of glucose and oxygen to yeast cells during storage time did appear to promote changes in kinetic activity, both detrimental and advantageous to industrial applications, which are identified in this study. The conclusions and observations made on this research have direct relevance to industrial applications and the development of predictable kinetic activity of yeast cells.

The facultative anerobic nature of <i>Escherichia coli</i> allows the study of the effects of oxygen on growth, metabolism and enzyme expression in a deliberately poorly mixed fermentation system that mimic conditions found in industrial fermenters. A methionine auxotroph of <i>Escherichia coli</i>, NCIMB 9481, grown on a defined medium, was used to investigate scale-down fermentations. Two approaches were taken to investigate this problem. The first involved the construction, characterisation and operation of a laboratory fermentation system. The continuous fermentation system consisted of a 2 L stirred tank reactor (CSTR) which is linked to a 0.5 L recirculating plug flow tubular reactor (PFTR). The second approach involved the development of a general mathematical model to describe the growth of <i>Escherichia coli</i> in the combined system (CSTR + PFTR). The model based on Monod kinetics, took into account the mixing environment in the reactor and was able to predict the oxygen sensitive substrate (glucose) consumption, endogenous metabolism, biomass formation, metabolic end-product formation (ethanol, acetic acid and carbon dioxide), and the expression of the malate dehydrogenase enzyme. The growth of <i>Escherichia coli</i> in the combined system and its physiological response to variations in recirculation rate, aeration rate, initial substrate concentration were then investigated. These results, when compared with those predicted by the simulation, demonstrated that the model gave an accurate prediction of microbial product formation with the exception of the metabolic end-products. Simulations were then used to explore the mixing characteristics of the combined system. In particular the response of the microbe to mixing as expressed by the volume fraction (εT), (V<SUB>PFTR</SUB>/(V<SUB>PFTR+</SUB> V<SUB>CSTR</SUB>)), and the recirculation ratio (R). The results show that εT significantly effects the performance of <i>Escherichia coli</i>. The recirculation ratios (R) only effect the system at high dilution rates where system deviates from CSTR mixing theory.

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Interferons have been recognized as important mediators of cellular communication for many years. There are two types of interferon: Type I interferons have antiviral functions, but Type II interferon (IFN-g) is more important as an immunomodulating molecule. Type II interferon has effects on cellular MHC class II expression, immunoglobulin class-switching, macrophage activation, cellular proliferation and a number of other functions. The role of IFN-g during <i>in vivo</i> immune responses has not been studied in great detail, but the sheep is an ideal species in which to study these phenomena by using the efferent lymphatic vessel cannulation model. This allows access to cells and tissue fluid for cytokine analysis using antibody and genetic probes for the detection of IFN-g. Bovine IFN-g peptides (amino-terminus, carboxy-terminus and central) were used to generate antibodies in rabbits. None of the anti-peptide sera reacted with denatured ovine or bovine IFN-g, nor neutralized their antiviral effect. Rabbit antibodies to bovine recombinant IFN-g neutralized ovine IFN-g and detected IFN-g in a sandwich ELISA when used in combination with a monoclonal antibody against a human IFN-g carboxy-terminal peptide. The sensitivity of detection was only 125ng/ml, insufficient for use with efferent lymph fluid samples. The expression of MHC class II molecules on cell surfaces is increased by IFN-g on many cell types. This has been used previously to measure biologically active IFN-g concentrations in fluids. Measurement of ovine class II by slot blot was assessed as a method of adapting this to ovine IFN-g measurement, but the technique proved to be too problematic for regular use. The expression of class II on T lymphocytes is influenced by IFN-g in the surrounding fluid. Analysis by FACS of resting ovine T lymphocytes shows them to express class II, a situation different to that in the human.