Avenell, Philip James; (2003) Downstream Processing from Baeyer-Villiger Monooxygenase Bioconversions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Although much work has been carried out into the study of bioconversions and the associated upstream process options, very little has focused on the issues of downstream product recovery. The overall process route to the purification of bioconversion products may be described in three parts: catalyst removal, product concentration and product isolation. This work aims to study the options for catalyst recovery and product concentration from a whole cell bioconversion. The model reaction for study is the commercially important bioconversion of racemic ketone bicyclo[3.2.0]hept-2-ene-6-one to the lactone products (1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (1R,5S)-3-oxabicyclo[3.3.0]-oct-6-en-2-one. This is achieved by the use of an Escherichia coli TOP 10 [pQR239] whole cell catalyst. Extraction of products was studied direct from the bioconversion in spent fermentation media and resuspended catalyst in phosphate buffer. Catalyst recovery was investigated by use of centrifugation and both micro- and ultrafiltration. Techniques applicable to the product concentration of the lactone products have been assessed. Only the use of physical adsorption and liquid-liquid extraction proved effective. The effects of process stream and choice of catalyst recovery technique on these concentration techniques has been studied. Physical adsorption was achieved by the use of the resin Amberlite XAD-4. This was successfully operated in a packed bed column producing an effective extraction and concentration step. The choice of primary recovery technique or operation of bioconversion had no effect on the column performance. However, it is necessary to remove the biocatalyst prior to loading in order to avoid a pressure increase across the column. The presence of substrate within the feed stream was shown to have a deleterious effect on column performance. This highlights the need for effective monitoring of the bioconversion in order to avoid significant residual substrate. The application of batch liquid-liquid extraction is poor due to the low partition coefficient of the products. Therefore, the use of a liquid-liquid continuous extractor was studied. This system is designed for use with low partition solvents and dilute concentration of components. The system has been demonstrated to be effective in the extraction and concentration of products from bioconversion media. An algorithm to describe the system was developed and the effect of the operating parameters on extraction rate established. The system is capable of extraction direct from the bioconversion although the process can not be agitated and operation is therefore slow. More rapid operation can be achieved by the application of a catalyst recovery step with the rate dependent on the level of agitation as determined by broth clarity. The highest clarity of bioconversion broth was achieved using ultrafiltration and this resulted in the shortest extraction time.

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