The dynamic influence of cells on the formation of stable emulsions in organic–aqueous biotransformations

Abstract

Emulsion stability plays a crucial role for mass transfer and downstream processing in organic-aqueous bioprocesses based on whole microbial cells. In this study, emulsion stability dynamics and the factors determining them during two-liquid phase biotransformation were investigated for stereoselective styrene epoxidation catalyzed by recombinant Escherichia coli. Upon organic phase addition, emulsion stability rapidly increased correlating with a loss of solubilized protein from the aqueous cultivation broth and the emergence of a hydrophobic cell fraction associated with the organic-aqueous interface. A novel phase inversion-based method was developed to isolate and analyze cellular material from the interface. In cell-free experiments, a similar loss of aqueous protein did not correlate with high emulsion stability, indicating that the observed particle-based emulsions arise from a convergence of factors related to cell density, protein adsorption, and bioreactor conditions. During styrene epoxidation, emulsion destabilization occurred correlating with product-induced cell toxification. For biphasic whole-cell biotransformations, this study indicates that control of aqueous protein concentrations and selective toxification of cells enables emulsion destabilization and emphasizes that biological factors and related dynamics must be considered in the design and modeling of respective upstream and especially downstream processes.

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

“…One of the identified proteins is the hydrophobic outer membrane protein A (OmpA) (P0A910, MOWSE Score 263.9, 4 peptides, 18.5% sequence coverage). Together with the finding that OmpA of different E. coli strains as well as the homologous protein AlnA of Acinetobacter radioresistens KA53 are secreted and act as emulsifiers (Walzer et al,), the presented results indicate that extracellular proteins play a decisive role not only in the stabilization of cell‐based emulsions (Collins et al,) but also in their scCO 2 ‐mediated irreversible phase separation.…”

“…As shown in the previous sections, changes in the particle (cell) properties due to toxification or cell disrupture can be excluded as causes for emulsion destabilization. In the accompanying study on emulsion stability during the biotransformation of styrene (Collins et al,), catastrophic phase inversion achieved via an increase of the organic/aqueous phase ratio was found to enable phase separation and the recovery of hydrophobic cells from the phase boundary. Thus, phase inversion was investigated as a possible physical effect responsible for scCO 2 ‐mediated phase separation.…”

“…Thus, phase inversion was investigated as a possible physical effect responsible for scCO 2 ‐mediated phase separation. Comparable to the addition of pure bis(2‐ethylhexyl) phthalate (BEHP) to trigger catastrophic phase inversion (Collins et al,), the added CO 2 largely partitions into and dissolves in the organic BEHP phase and thus increases the organic/aqueous phase ratio. This change in phase ratio may lead to phase inversion and detachment of particles from the interface resulting in emulsion destabilization.…”

“…From a biological point of view, it is unlikely that such an immediate change in surface hydrophobicity has physiological causes. Collins et al () showed that, upon introduction of the organic phase, the concentrations of several proteins in the aqueous phase significantly decrease. These proteins may adsorb to the aqueous/organic interface and/or onto the cells’ surfaces increasing the surface hydrophobicity of the cells.…”

“…The treatment of the cells preferentially residing at the interface with HIC‐desorbents led to a decrease in the apparent cell‐surface hydrophobicity as also shown for the treatment with scCO 2 . After subsequent centrifugation, the supernatant contained the protein fractions of which the aqueous concentration had decreased upon organic phase addition (Collins et al (). These results indicate that the irreversibility of phase separation by means of scCO 2 is linked to the desorption of proteins from the cell surface or the aqueous‐organic interface.…”