Osborne, Stephen John; (1990) A study of the factors influencing the stability and operation of cellular biocatalysts in the presence of organic solvents. Doctoral thesis (Ph.D.), University College London.

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Abstract

The microbial conversion of compounds that have a low solubility in aqueous systems is typified by the fungal hydroxylation of steroids. In systems of this type the substrate of the reaction is of very low water solubility causing mass transfer limitations. Incorporation of a second organic liquid phase, in which the substrate has a far greater solubility, may be used to overcome such problems. Using the 11α-hydroxylation of the steroid progesterone by the fungus Rhizopus nigricans as a model system, a packed-bed design of bioreactor is assessed with respect to its suitability for use in conjunction with an organic-aqueous two liquid-phase system. The fungal biocatalyst was not immobilised but packed as whole cells, either filamentous or pelletted, into the column reactor. It was considered that the packed-bed might act as a static mixing device so maintaining an emulsion on the bed. Results show the unsuitability of operation of the reactor with an emulsion or an alternative single organic phase. However, use of a methanol/aqueous cosolvent is shown to produce a comaratively stable bed from which 11α-hydroxylase activity data was obtained. A detailed study is presented of the causes for the loss of 11α-hydroxylase activity by cells of R.nigricans in systems containing an organic liquid phase. It is shown that a strong correlation exists between the loss of 11α-hydroxylase activity and the concentration of organic solvent that partitions into the cell membranes. The concentration of organic solvent present in the membranes may be calculated from a modification of a Collander type equation. For R.nigricans in the particular aqueous buffer system employed this becomes [solventmembrane] = 0.19 x Poctanol x [solventaqueous] is the concentration of organic solvent partitioning into the membranes, [solventmembrane] is the concentration of organic solvent dissolved in the aqueous phase and Poctanol partition coefficient for the organic solvent in a standard octanol/aqueous system. Total loss of activity is shown to occur at a single critical membrane solvent concentration irrespective of the organic solvent type. Organic solvents unable to achieve this critical membrane concentration are shown to allow the retention of hydroxylase activity at saturating aqueous phase concentrations. However, with the development of a discrete second organic liquid phase loss of activity occurs through so termed 'phase' effects. Design parameters are given, based on the above criteria, that will aid the rational selection of organic solvents for future two liquid-phase reactor systems.

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