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No description available.

Several continuous flow systems utilising reactors containing soluble enzymes have been designed and built. Various reactor configurations have been examined using the urease catalysed hydrolysis of urea as a model. Preliminary experiments showed that the attainment of a steady state within a continuous stirred tank reactor was dependent on a number of environmental conditions. Loss of enzyme activity was partially prevented by using EDTA in the inlet feed. However enzyme activity was also partially lost in a thin channel reactor by shear forces and adsorption of the enzyme onto the membrane surface. Studies on the enzyme suggest that the shear effect is reversable. Analysis of the residence time distributions in a thin channel reactor showed that under certain controlled conditions this reactor could be considered as an ideally-mixed vessel. Kinetic parameters obtained using this assumption agreed well with those obtained from steady state kinetics. The effect of flow rate on conversion and the minimising of substrate inhibition also indicated a similarity between this reactor and an ideally-mixed vessel. The possibility of using glycerol kinase within a thin channel reactor to produce sn-glycerol 3-phosphate was investigated. Results indicated that reactor operation at the pH optimum of the enzyme did not produce the greatest enzyme stability. Carbamate kinase v/as successfully used within the reactor to reconvert the ADP produced by glycerol kinase back to ATP, thus increasing the efficiency of cofactor utilisation. To further increase the efficiency of ATP usage the cofactor was successfully immobilized to a soluble high molecular weight dextran by a new facile method so that ATP would be retained within the reactor. The immobilized cofactor acted as a substrate for glycerol kinase and several other kinases. However it was not active with carbamate kinase obtained from Streptococcus faecalis is R, and thus could not be used profitably within the reactor.

The aim of this work was to investigate the production of antimicrobial compounds by marine epiphytic bacteria. This work was carried out by devising a "niche mimic bioreactor", in which physical and chemical conditions were similar to the original ecological niche from which the bacteria were isolated. A modified roller bottle bioreactor was designed to mimic the intertidal environment and could facilitate the production of antimicrobial compounds by two marine isolates. Biofilm formation by these bacteria was found to be the key requirement for this observation. An Air- Membrane Surface (AMS) bioreactor was therefore constructed to allow growth of attached bacteria within a biofilm. A Bacillus licheniformis strain, EI-34-6, isolated from the surface of the marine alga Palmaria palmala, produced bacitracin and a red pigment when cultivated using the AMS bioreactor but not using standard shake flask cultures. Glycerol and ferric iron were necessary for the production of both antimicrobial compounds and the red pigment. Further investigation into the mechanism of this induction showed that a small amount of the cell-free spent medium from the AMS culture of this isolate could induce the corresponding shake flask culture to produce bacitracin and the red pigment. Glycerol or ferric iron was not necessary for the production of inducer compounds. Furthermore, metabolites from the shake flask culture, which had been induced to produce bacitracin and the red pigment, were able to continue to induce other non-producing shake flask cultures. In addition, a small amount of cell-free spent medium from B. subtilis DSMIOT grown using the AMS bioreactor also induced B. licheniformis strain EI-34-6 to produce bacitracin and the red pigment in shake flask cultures. However, the spent medium from B. subtilis DSMIOT shake flask culture could not elicit this production. These results suggest the presence of a biofilm specific and cross-species signalling system which can elicit, in planktonic bacterial cells, the type of metabolism normally observed in cells grown in a biofilm. Based on this discovery, the AMS bioreactor was improved and it allowed a further nine Bacillus isolates to be obtained, which exhibited a similar phenomenon. The observations suggest a novel strategy to discover new antimicrobial compounds by taking advantage of their signalling system to reveal new metabolic pathways in marine microorganisms

An industrial-type fermentation for the production of alphaamylase was studied at 3-litre scale, using a complex medium and Bacillus subtilis B. 20, a high-yielding strain. It was found that both the peak titre and the production rate continued to increase up to the highest mass-transfer rates used. Using an impeller of half the fermenter diameter at speeds around 14 Hz (840 r. p. m. ) and aeration at 0.57 v. v. m., a batch fermentation of 60-70 hours gave at least 9 u/ml (about 900 S. K. B. u/ml, measured at pH 6.0, or 36 000 Novo u/ml). Rapid production began around hour 10, when growth was about half complete. The volumetric production rate was almost constant for 40-50 hours, giving a constant specific productivity of about 0.75 x 10-11 u/cell/h after the completion of growth, until an abrupt final decline. Rapid synthesis coincided with growth and the rate was not directly related to population, the rates of growth and mass-transfer or any other measured variable. The termination of synthesis in simple batch operation was apparently due to carbohydrate depletion. Thus, feeding increased production by about 25%. Corrected for dilution, the production rate was unchanged so that the specific productivity fell in proportion to the increase in population during feeding. It is suggested that the rate of synthesis was controlled by a pool of a very stable regulator-inducer complex accumulated before exhaustion of the inducer in the early stages of the fermentation. There appears to be considerable potential for further development along the lines followed in this work. The limits of the stimulatory effects of mass transfer and feeding were not passed. Neither was there evidence of depletion of a pool of complexed regulator.

Although L-glutamate production by Corynebacteria fermentation has been well-established in the biotechnology industry since 1960’s, the demand for this building block for human consumption is increasing enormously. Researchers have therefore been trying to improve the yield and productivity of amino acids by developing genetically modified organisms or through the use of metabolic engineering tools. In this study, a new approach by which the utilization of transient electropermeabilization for increasing the membrane permeability of these bacteria to L-glutamate secretion will be investigated, thus potentially enhancing the yield of glutamic acid production. Micrococcus glutamicus (a strain of Corynebacteria) cultivated in CGXII Minimal Medium was successfully used to produce L-glutamate under different growth conditions i.e., biotin limitation, surfactant (Tween 40) addition and ethambutol addition. This study clearly showed that the production of L-glutamate is influenced by the concentration of these agents and by their time of addition into the fermentation medium. The highest amount of L-glutamate was found to be 57mM at a biotin concentration of 1µg l-1. A simple method based on centrifugation and acid-base addition was developed in which L-glutamate could be separated with a purity of 90% from the fermentation broth. This research confirmed that cell growth, viability, substrate consumption and L-glutamate production by M. glutamicus are notably affected by the increase of medium osmolality (caused by the addition of NaCl). This phenomenon is often observed because of the use of high concentration industrial medium or accumulating amino acids into the extracellular medium during fermentation. This study confirmed that the conductivity of medium in which cells are suspended represents a major barrier for excreting the intracellular protein or enzyme by membrane electropermeabilization, regardless of the electric field strength and the number of pulses applied. The electroenhancement of L-glutamate secretion by the above-mentioned treatments is limited due to the high conductivity of fermentation broth. However, an increase (about 19%) of total protein excreted from M. glutamicus as compared to the control was obtained in cells suspended in a less conductive medium (dH2O) and electroporated at 12.5kV cm-1, 200 and 25µF with 3 pulses. It was also observed that electroporation factors, especially field strengths, pulse numbers, medium conductivity and the configuration of cells have a major impact on cell viability. The yield of electrosecretion will be limited due to the reduction in cell viability during continuous microbial fermentation and metabolite extraction by the high intensity electric pulses used. This research has established many of the factors requiring consideration when using electroporation for bioprocess intensification.

No description available.

No description available.

No description available.