Replacement of a considerable part of the traditional, aqueous reaction medium in biotechnology by an organic medium is a promising technique to broaden the scope and range of biotechnological processes. This seems especially to be true for the conversion of non-polar substances. The high capacity of solvents for sparingly water-soluble substrates and products could reduce the required volume of the reaction mixture significantly, and may also lead to less substrate and/or product inhibition in the aqueous biocatalyst phase, when these mechanisms are involved. Furthermore, the use of an organic solvent could shift reaction equilibria favourably and facilitate down-stream processing. In chapter 1 a general review is presented of non-aqueous solvent systems in biocatalytic processes. Special attention is paid to two-liquid-phase systems, involving water-immiscible solvents. Several facets of these biphasic systems have been studied in this thesis using the epoxidation of propene by gel-entrapped <em>Mycobacterium</em> cells as a model.<p>After the description of the throughout this work employed techniques of gas-analysis automatization and of substrate-level control (chapter 2), the far-reaching consequences of the solvent choice are treated in chapter 3. Many solvents cause rapid inactivation of the free, propene-epoxidizing cells. This appears also to be the case if the cells are immobilized in calcium alginate. However, the support material prevents direct cell-organic solvent contact and the associated aggregation and clotting of cells, mostly accompanied with loss of activity. High activity retentions of the immobilized cells relate to low polarities and high molecular weights of the used solvents. The polarity, as expressed by the Hildebrand solubility parameter, is also useful for describing the solvent capacity for one of the two substrates, oxygen, and for the product, propene oxide. The capacity for propene is less well described by the Hildebrand solubility parameter, but also less relevant, as the capacity of the solvents for propene is always about two orders of magnitude higher than that of water, and thus limitation of the rate by unsufficient supply of propene is less likely to occur. It is stressed that optimization of the solvent polarity is necessary, as the requirement of a high activity retention conflicts with the need for a high solvent capacity for the polar propene oxide. Optimization of the polarity will also be likely in case of other types of two-liquid-phase bioconversions.<p>External and internal-diffusion limitations, which are to be expected when using cells entrapped in a hydrophilic: gel, are quantified in chapters 4 and 5. With negligible product inhibition, satisfactory predictions of the mass-transfer effects on the intrinsic Michaelis-Menten kinetics of the immobilized cells are obtained by using a simple pore-diffusion model (chapter 4). Internal diffusion is found to severely limit the epoxidation rate. A more complex model for the intrinsic epoxidation kinetics has been derived for modelling of mass-transfer rates in case of product inhibition (chapter 5).<p>The microkinetic model defined in chapter 4 is integrated in a macrokinetic model to describe the behaviour of a packed-bed immobilized-cell reactor (chapter 6). Depletion of the limiting substrate, oxygen, along the length of the bioreactor can be prevented by using an organic solvent, n-hexadecane, as the transport medium. It is argued that this finding may eliminate the need for a separate gas phase in the fixed-bed reactor. Model predictions of the oxygen conversion in the bioreactor at various degrees of external and internal-diffusion limitation, at various liquid space times and with water or n-hexadecane as the continuous phase are in good agreement with experimentally obtained values. In chapter 7 some other, main limitations of the epoxide production in the packed-bed organic-liquid-phase/immobilized-cell reactor are quantified. Product inhibition is reduced by absorption of the inhibitory epoxide in a cold di-n-octyl phthalate phase. The stability of the immobilized cells is increased by supplying the cells alternately with propene and a co-substrate (ethene). About 50 g dry weight of cells in a 1.7 dm <sup><font size="-1">3</font></SUP>packed-bed reactor were used, which produced ~ 1.5 g chiral propene oxide; two third of the epoxide was absorbed in the octyl phthalate phase.<p>Finally, in the last chapter of this thesis a general discussion is presented. The significance of optimization of the solvent polarity and of the interphase polarity, i.e. the polarity of the phase between biocatalyst and organic solvent is underlined. In case of entrapment in prepolymers, the hydrophobicity/hydrophilicity balance of the gel can be optimized with respect to polarities of substrates and products. Several features of hydrophilic and hydrophobic gels are compared. A quantitative illustration is given concerning the design on a technical scale of a fixed-bed organic-liquid-phase/immobilized-cell reactor. The advantages of using solvents with a high substrate capacity (often oxygen in case of aerobic processes) are demonstrated.
|Qualification||Doctor of Philosophy|
|Award date||5 Nov 1986|
|Place of Publication||Wageningen|
|Publication status||Published - 1986|
- chemical industry
- chemical reactions