The utilisation of a biological catalyst to selectively transform a substrate molecule to a product is established in the literature from early fermentation processes such as the modem manufacture of antibiotics and other fine chemicals. However, many potently substrate/product molecules are poorly water soluble or ^ e toxic to the biocatalyst. Thus a process for the conversion of these substrates is inefficient and problematic (Lilly and Woodley, 1985). A potential solution to overcome poor aqueous solubility and/or toxicity is to dissolve the substrate in an organic solvent as a second phase in the reactor. This enables a constant supply o f substrate to be made available to the biocatalyst and also enables a degree of control over the delivery of toxic substrate into the vicinity o f the biocatalyst.
The use of an organic solvent as a substrate and product reservoir for two-liquid phase biotransformation was first reported in the 1930s (Hailing and Kvittingen, 1999). However, the potential of this technology was not fully realised at the time. In the early 1970s the technology was ‘rediscovered’, being initially used in the biotransformation of poorly water soluble steroids (Cremonesi et a l, 1975), and it was rapidly adapted for use with other poorly water soluble molecules (Buckland et a l, 1975; Klibanov et ah, 1975).
The use of organic solvents in bioreactors has been reviewed extensively e.g. Cabral et a l, 1997; Eggers et a l, 1989; Fernandes et a l, 1995; Woodley et a l, 1991; Lye and Woodley, 2001. There are several major advantages to their application in a two-liquid phase system:
• Increased solubility o f substrate and product. If a substrate/product is poorly water soluble dissolving it in solvent can make a process economically viable. Due to an increase in the availability of the substrate for the biocatalyst or extraction of the product i.e. use of organic solvent enabled dehydrogenation of the water-insoluble 6-a-methyl-hydro-cortisone-21-acetate by Arthrobacter simplex cells (Fernandes et a l, 1995).
• Alteration of enzyme selectivity, Placing an enzyme in nearly anhydrous media can change the selectivity o f the enzyme (Carrea et a l, 1995).
• Reduce product inhibition. Inhibition of a biocatalyst can be reduced by the extraction o f the product from the aqueous phase.
• Shift reaction equilibria. Transfer of products to the organic phase can shift the apparent reaction equilibria.
• Product recovery. As the solvent may contain a higher product concentration and different physical properties compared to water this can facilitate improved product recovery by separation of the two phases and distillation of the product containing phase.
A measure o f a solute affinity for a particular solvent is the distribution coefficient. This is defined as the ratio of the solute concentration in the solvent to the solute concentration in the aqueous medium at equilibrium (Bruce and Daugulis, 1991). Therefore, it follows that as the distribution coefficient increases, the greater the affinity o f the substrate/product molecule for the solvent. Using the distribution coefficient workers can therefore determine whether a particular solvent will be useful for delivering substrate to the aqueous phase or whether it will be useful in extracting product from the aqueous phase.
The use o f organic solvents can also has potential disadvantages within a two- phase bioreactor such as inhibition or inactivation of the biocatalyst due to interfacial effects and molecular toxicity of the solvent. In extractive fermentations, unintentional formation of emulsions and protein stripping can also occur (Van Sonsbeek et a l, 1993). Therefore, an important consideration in the design o f a two-liquid phase biotransformation is the relationship between the solvent and biocatalyst. This depends on the choice of solvent and the form of the catalyst.
1.4.1 Catalyst form
For a given two-phase biotransformation a biocatalyst can be applied in several forms, either as a whole cell, a ffee/immobilised isolated enzyme or as a homogenous cell extract. The choice of biocatalyst form depends on many factors
the enzyme, whether or not the enzyme can be isolated, the type of reaction and the reaction conditions e.g. T °C (Faber, 1997a).
Generally whole cell catalysts are easy to prepare from a fermentation and can therefore be produced rapidly and cheaply. Whole cell catalysts tend to be more stable than isolated enzymes due to the presence of a cell membrane/wall (Woodley et al., 1990). However, side reactions on the substrate may occur inside the cell (Tramper, 1996) and larger substrates will not be easily transported across the cells membrane. Also, the physiology of the cell can lead to further process issues such as surfactant release (schmid et al., 1998) and increased viscosity of the aqueous phase. Enzymes are more expensive to prepare but are unlikely to produce side reactions and are able to interact with larger substrates. They can also be used in near anhydrous conditions where reactions can be reversed such as deyhdration instead o f hydrolysis (Westcott and Klibanov, 1994; Carrea et al.,
1995; Layh and Willetts, 1998).
1.4.2 Immobilisation of the biocatalyst
The stability and recovery of the different forms o f biocatalyst, particularly for isolated enzymes, can be improved by immobilisation. Immobilisation protects the enzyme/cell by making it more difficult to unfold or disrupt by fixing it to a support usually a polymer. The various methods o f immobilisation have been extensively reviewed and will not be described here (Ballesteros et al, 1994). Other advantages of immobilisation include an increase in productivity. As well as facilitated recovery o f the biocatalyst immobilisation can also protect the biocatalyst against damage from the reactor itself, such as shearing effects and reduce aggregation o f biomass at the liquid-liquid interface (Brink and Tramper, 1985; Van Sonsbeek et al., 1993). However, Brink and Tramper also observed that this did not reduce solvent molecular toxicity effects on the catalyst.