A Comparison of High Cell Density Fed-Batch Fermentations Involving Recombinant Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum Under Well-Mixed Small-Scale and Simulated Poorly Mixed Large-Scale Conditions

In this work, multi-parameter flow cytometry, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of recombinant protein production in high cell density fed-batch cultures of Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. Further, we report on the development of the scale-down, two compartment (STR-PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on 3 industrially relevant recombinant bacterial systems. Using various scale-down configurations, it has been shown that recombinant protein production can be followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on the organism studied and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.