Applied catastrophic phase inversion: a continuous non-centrifugal phase separation step in biphasic whole-cell biocatalysis.

Biphasic whole-cell biotransformations are known to be efficient alternatives to common chemical synthesis routes, especially for the production of, e.g. apolar enantiopure organic compounds. They provide high stereoselectivity combined with high product concentrations owing to the presence of an organic phase serving as substrate reservoir and product sink. Industrial implementation suffers from the formation of stable Pickering emulsions caused by the presence of cells. State-of-the-art downstream processing includes inefficient strategies such as excessive centrifugation, use of de-emulsifiers or thermal stress. In contrast, using the catastrophic phase inversion (CPI) phenomenon (sudden switch of emulsion type caused by addition of dispersed phase), Pickering-type emulsions can be destabilized efficiently. Within this work a model system using bis(2-ethylhexyl) phthalate (BEHP) as organic phase in combination with E. coli, JM101 was successfully separated using a continuous mixer settler setup. Compared to the state-of-the-art centrifugal separations, this process allows complete phase separation with no detectable water content or cells in the organic phase with no utilities/additives required. Furthermore, the concentration of the product is not affected by the separation. It is therefore a simple applicable method that can be used for separation of stable Pickering-type emulsions based on the knowledge of the point of inversion.

Process boundaries of irreversible scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.