Monoclonal antibodies can be produced in culture fluid by the fermentation of specificially selected hybridoma cells. Hybridoma cells exhibit suspension type fermentation characteristics and therefore the simplest method for large scale fermentation is that of the stirred tank fermenter. However, such is the growing demand for monoclonal antibodies, methods for increasing the production capacity of a commercial process are being developed. This study examines some of the current process intensification methods in relation to an established production facility. As well as examining the actual productivity increases possible with any method, the applicability of that method to a commercial environment is taken into account. Hollow-fibre systems are investigated, with a potential increase in productivity which was outweighed by the significant retooling and retraining costs. Gel Bead entrapment systems are shown to have great promise, as they can be readily placed into existing equipment and production methods. However, all methods examined, including alginate bead entrapment, were found unsuitable for hybridoma cell culture. A novel method for cell entrapment was developed, using an agarose/alginate gel mixture which allowed greatly improved growth and consistent antibody production. The entrapment method was examined in a continuous chemostatic system. This system was then scaled-up and applied to the existing facility, to give a 25L airlift operating in a chemostatic mode at a rate of 1.2-1.5 day<SUP>-1</SUP>.

The present study describes physiological conditions which promote curdlan synthesis, the effect of metabolic inhibitors on production and the nature of the product of <I>Agrobacterium </I>sp. ATCC 31749, a laboratory derivative of a strain originally isolated from soil. Nitrogen depletion in the medium was essential for production, and depletion of sulphur or phosphorus in batch culture did not promote curdlan synthesis. Similarly, initiation of curdlan production was observed when bacteria were transferred to nitrogen free medium but not media free of sulphur or phosphorus. A model of the mechanism of nitrogen dependent control is presented. <I>Agrobacterium </I>sp. ATCC 31749 grew well on a number of monosaccharides and lactose, moderately well on succinate and poorly on glycerol. Good curdlan production was observed from mannose, glucose and galactose with a reasonable curdlan yield obtained from sucrose. Curdlan yields when lactose, maltose or glycerol were supplied were poor and no curdlan was obtained from culture on succinate. The effects of other physiological conditions were examined. Arsenate inhibited neither initiation nor continuation of curdlan biosynthesis when added to the medium in concentrations up to 10mM. 5mM sodium azide inhibited curdlan production but not glucose uptake. A similar effect was observed when the ionophore tetracaine (1mgml<SUP>-1</SUP>) was added to the medium. EDTA inhibited curdlan production and glucose uptake whilst EGTA inhibited neither. Chloramphenicol inhibited curdlan production as did rifampicin. Analysis of the kinetics and degradation products of enzyme hydrolysis revealed a similarity between neutralised gels and gels formed by low temperature heating. Results obtained with native curdlan or curdlan preparations obtained by boiling aqueous suspensions were similar. The implications or these results on curdlan biosynthesis are discussed.

A sensor exploiting the properties of Green Fluorescent Protein (GFP) was developed and was used to optimise fennentation conditions for production of the soluble fusion protein CheYGFP. A 9-fold increase in yield was achieved. Genes encoding CheY, Defl and DXR were fused in-frame to the 5' and 3' end ofthe GFP gene to detennine whether this affected optimised production of soluble GFP fusion proteins. Different conditions were required for optimal accumulation of the recombinant protein and the optimised conditions for CheYGFP and DXRGFP were identical to those for CheY and DXR respectively. Conditions optimal for the accumulation of Defl, DeflGFP, GFPDefl, GFPCheY, GFPDXR and GFP were the same. During several fennentations the production of soluble recombinant protein did not increase after 12 h. This was concomitant with a rapid increase in the rate of carbon dio~ide production by the culture. Cell pellets after centrifugation contained a layer of bacteria that were not fluorescent, indicating the presence of a non-productive population of bacteria. Restriction mapping and sequence analysis confinned the presence after 12 h of bacteria carrying a plasmid in which a frameshift mutation had occurred. Improvement in the maintenance of the original plasmid was achieved by further manipulation offennentation conditions.

The application of novel emulsification technologies was investigated in order to improve the utilisation of oil, the production of oxytetracycline and the viscosity during industrial and non-industrial Streptomyces rimosus fermentations. Emulsifiers (Mergital Q3V® and Agnique RSO 10®) mixed with rapeseed oil were fed to industrial S. rimosus fermentations. In addition, the Phase Inversion Temperature technology (PIT rapeseed and sunflower oil emulsions) was applied on both industrial and non-industrial S. rimosus fermentations. Similarly, the Self-Emulsifying technology was used for the production of a rapeseed oil feed (Agnique RSO 3®) which contained a hydrophilic group and was fed to S. rimosus industrial and non-industrial fermentations. Increased oil utilisation was recorded in the above processes. A very significant decrease (55-65%) in the final concentration of residual oil was achieved when PIT and self-emulsifying technologies were used. A significant decrease (50-75%) in the viscosity was also observed in the self-emulsifying process. It was concluded that the increase in the utilisation of oil was due to the smaller and stable oil droplet size offered by such technologies and/or the lower viscosity which increased the mass transfer from the complex fermentation medium to the cells. The final antibiotic (oxytetracycline) concentration did not significantly increase in any of the industrial and non-industrial fermentations. However, the microorganisms was able to produce oxytetracycline after oil feed termination when oil emulsions were used to contrast to normal rapeseed oil where no production was observed. Morphological analysis of the dispersed mycelia was carried out providing information on the length and number of tips of the free mycelia. When oil was utilised at higher rates, the microorganisms showed higher metabolic activity which led to longer filaments. This was probably due to better carbon availability increasing production rate of elongation precursors inside the mycelia.

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The aims of this project are to better understand the biosynthesis of CDA and then use that knowledge to genetically manipulate Streptomyces to produce novel compounds. The natural structural variants of CDA which have been characterised contain several unusual amino acids residues. CDA contains both D-figured and non-proteinogenic residues within its structure. In all of the CDA structures isolated so far, position six is occupied by a D-figured hydroxyphenylglycine residue (D-4-HPG). The precursor biosynthetic pathway for HPG has been elucidated in A. orientalis and it has been shown previously that the cda cluster contains homologues to these genes. A mutant strain of S. coelicolor in which the hmaS (4-hydroxymandelic acid synthase) gene had been disrupted had previously been created and shown to be deficient in CDA production. During this study the proposed intermediates of the HPG biosynthetic pathway were fed into the mutant and CDA production re-established thus proving the pathway for HPG biosynthesis in S. coelicolor mirrors that seen in other organisms. Feeding analogues of the pathway intermediates to the mutants resulted in the mutasynthesis of novel lipopeptides with modified arylglycine residues. This study also identified a gene from the cda cluster, hasP, which in silico analysis predicted to encode a 3-hydroxyasparaginyl phosphotransferase responsible for the biosynthesis of 3-phosphohydroxyasparagine sometimes found at position nine in the CDA structure. Deletion of the majority of the gene resulted in only non-phosphorylated CDA variants being produced.

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The depletion of non-renewable energy resources, the environmental concern over the burning of fossil fuels, and the recent price rises and instability in the international oil markets have all combined to stimulate interest in the use of fermentation processes for the production of alternative bio-fuels. As a fuel, ethanol is mainly of interest as a petrol additive, or substrate, because ethanol-blended fuel produces a cleaner, more complete combustion that reduces greenhouse gas and toxic emissions. As a consequence of the surge in demand for biofuels, ethanol producing microorganisms, such as the bacterium Zymomonas mobilis, are of considerable interest due to their potential for industrial-scale bioethanol production. Although bioethanol has traditionally been produced in batch fermentation with the yeast Saccharomyces cerevisiae, there are advantages in using Z. mobilis as an alternative for bioethanol production. In comparison to yeast, Z. mobilis grows and ferments rapidly, without the requirement for the controlled supply of oxygen during fermentation, and has a significantly higher ethanol product rate and yield. Most importantly, it has a high tolerance for ethanol. Bacteria communicate with one another using chemical signalling molecules. In general, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules termed autoinducers (AI). This process allows bacteria to monitor the environment for other bacteria and to alter behaviour on a population-wide scale in response to changes in the number and/or species present in a community. Currently, there are three well-defined classes of molecules that serve as the paradigms for chemical signaling in bacteria: oligopeptides, AI-1 (AHLs) and AI-2. Oligopeptide signalling is the predominant signal used by Gram-positive bacteria, and AHLs (acyl-homoserine lactones) are for species-specific communication in Gram-negative bacteria. Finally, the LuxS/AI-2 pathway is generally considered as involved in interspecies communication because the luxS gene, which is responsible for AI-2 production, is found in various bacteria. Many physiological functions in bacteria such as toxin, virulence factor and bacteriocin production, biofilm formation, bioluminescence, type III secretion, have been shown to be under the control of AI-2 quorum sensing. In Z. mobilis, in vitro synthesized and in vivo produced AI-2 treatment enhanced ethanol production by this bacterium up to a maximum of 50% in comparison with untreated control cells. This appears attributable to the overproduction of the glycolytic enzymes, enolase and pyruvate carboxylase, which are only rarely found in bacteria and the key enzymes for ethanol production. From the perspective of interspecies communication, enhanced ethanol production in Z. mobilis, under the control of the AI-2 signalling molecules, could represent a good example of a bacterium that does not produce AI-2, but responds to it. Another interesting finding is that two extracellular proteins from Z. mobilis, ZMO0994 and ZMO0134 which were originally induced by AI-2, were secreted when they were cloned, transformed and expressed in E. coli strain BL21 DE3; since it is generally accepted that nonpathogenic strains of E. coli, particularly derivatives of K12, do not secrete proteins under routine growth conditions. Presumably, these proteins possess signal sequences for secretion that could be used to provide a strategy for their use as carriers of recombinant proteins produced in E. coli K12. The merit of this system is that there would be few contaminant cytoplasmic proteins, and could possibly solve problems in protein purification, such as protease activity, protein misfolding and inclusion body formation. Finally, the discovery that the metabolic pathway leading to ethanol production is regulated by AI-2 is of considerable biotechnological importance because it will provide a basis for further engineering of strains for more efficient ethanol production. Indeed, engineering Z. mobilis by introducing the genes that encode Pfs and LuxS to produce AI-2, would be a means to stimulate increased ethanol production.