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. Biotechnol. Bioeng. 2015;112: 2316–2323. © 2015 Wiley Periodicals, Inc.

[1]  D. Fraser Bursting Bacteria by Release of Gas Pressure , 1951, Nature.

[2]  Mukul M. Sharma,et al.  Factors Controlling the Stability of Colloid-Stabilized Emulsions: I. An Experimental Investigation , 1995 .

[3]  Y. Pyun,et al.  Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment. , 2001, International journal of food microbiology.

[4]  R. Carbonell,et al.  A Novel Process for Demulsification of Water-in-Crude Oil Emulsions by Dense Carbon Dioxide , 2003 .

[5]  M G Wubbolts,et al.  Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase. , 2000, Biotechnology and bioengineering.

[6]  J. Woodley,et al.  Towards large-scale synthetic applications of Baeyer-Villiger monooxygenases. , 2003, Trends in biotechnology.

[7]  Andreas Schmid,et al.  Efficient phase separation and product recovery in organic‐aqueous bioprocessing using supercritical carbon dioxide , 2010, Biotechnology and bioengineering.

[8]  H. H. Beeftink,et al.  Two-liquid-phase bioreactors. , 1993, Enzyme and microbial technology.

[9]  S. O. Lumsdon,et al.  Catastrophic Phase Inversion of Water-in-Oil Emulsions Stabilized by Hydrophobic Silica , 2000 .

[10]  Masayuki Taniguchi,et al.  Sterilization of Microorganisms with Supercritical Carbon Dioxide , 1987 .

[11]  R. Dik,et al.  INACTIVATION OF MICROORGANISMS BY CARBON DIOXIDE UNDER PRESSURE , 1989 .

[12]  Andreas Schmid,et al.  Practical issues in the application of oxygenases. , 2003, Trends in biotechnology.

[13]  F. Hsieh,et al.  MICROBIAL SAFETY OF SUPERCRITICAL CARBON DIOXIDE PROCESSES1 , 1998 .

[14]  A. Schmid,et al.  Process implementation aspects for biocatalytic hydrocarbon oxyfunctionalization. , 2004, Journal of biotechnology.

[15]  F. Braet,et al.  Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells , 1997, Journal of microscopy.

[16]  T. Hashimoto,et al.  Inactivation of food microorganisms by high-pressure carbon dioxide treatment with or without explosive decompression. , 1997, Bioscience, biotechnology, and biochemistry.

[17]  Bernhard Hauer,et al.  Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis , 2002, Applied and Environmental Microbiology.

[18]  L. Garwin,et al.  ROSE PROCESS IMPROVES RESID FEED , 1976 .

[19]  Mattijs K. Julsing,et al.  Systematic optimization of a biocatalytic two-liquid phase oxyfunctionalization process guided by ecological and economic assessment , 2012 .

[20]  E. Ron,et al.  The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier. , 2006, Environmental microbiology.

[21]  U von Stockar,et al.  The influence of pressure and temperature of compressed CO2 on the survival of yeast cells. , 1995, Journal of biotechnology.

[22]  Andreas Schmid,et al.  Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase. , 2002, Biotechnology and bioengineering.

[23]  P. Fernandes,et al.  Whole-cell biocatalysis in organic media , 1998 .

[24]  O. Erkmen,et al.  High carbon dioxide pressure inactivation kinetics of Escherichia coli in broth , 2001 .

[25]  Andreas Schmid,et al.  The dynamic influence of cells on the formation of stable emulsions in organic–aqueous biotransformations , 2015, Journal of Industrial Microbiology & Biotechnology.

[26]  Daniel Kuhn,et al.  Intensification and economic and ecological assessment of a biocatalytic oxyfunctionalization process , 2010 .

[27]  D. Wasan,et al.  A possible mechanism of stabilization of emulsions by solid particles , 1992 .

[28]  A. Schmid,et al.  Integrated organic–aqueous biocatalysis and product recovery for quinaldine hydroxylation catalyzed by living recombinant Pseudomonas putida , 2012, Journal of Industrial Microbiology & Biotechnology.

[29]  M. Lilly Two‐liquid‐phase biocatalytic reactions , 2007 .

[30]  D B Kell,et al.  Solvent selection for whole cell biotransformations in organic media. , 1995, Critical reviews in biotechnology.

[31]  J. Sjöblom,et al.  The Role of Asphaltenes in Stabilizing Water-in-Crude Oil Emulsions , 2007 .

[32]  J. Cuq,et al.  Inactivation of Escherichia coli by carbon dioxide under pressure , 1996 .

[33]  Mukul M. Sharma,et al.  Factors controlling the stability of colloid-stabilized emulsions. IV. evaluating the effectiveness of demulsifiers , 1995 .

[34]  Inge Harald Auflem,et al.  Influence of pressure and solvency on the separation of water-in-crude-oil emulsions from the North Sea , 2001 .

[35]  D. Hopwood,et al.  Fixatives and fixation: a review , 1969, The Histochemical Journal.

[36]  Y. Pyun,et al.  Inactivation Kinetics of Lactobacillus plantarum by High Pressure Carbon Dioxide , 1999 .

[37]  O. Erkmen Effects of high-pressure carbon dioxide on Escherichia coli in nutrient broth and milk. , 2001, International journal of food microbiology.

[38]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[39]  Ho-mu Lin,et al.  An Improved Method for Disruption of Microbial Cells with Pressurized Carbon Dioxide , 1992, Biotechnology progress.