Studies related to the scale-up of high-cell-density E-coli fed-batch fermentations using multiparameter flow cytometry: Effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration

Multiparameter flow cytometric techniques developed in our laboratories hav e been used for the "at-line" study of fed-batch bacterial fermentations. T hese fermentations were done at two scales, production (20 m(3)) and bench (5 x 10(-3) m(3)). In addition, at the bench scale, experiments were undert aken where the difficulty of achieving good mixing (broth homogeneity), sim ilar to that found at the production scale, was simulated by using a two-co mpartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progr essive change in cell physiological state generally occurs throughout the c ourse of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and diss olved oxygen tension [DOT]) has a profound effect on cell physiology and he nce on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermenter were found to lead to a low biomass yield, but, surpr isingly, were associated with a high cell viability (with respect to cytopl asmic membrane permeability) throughout the fermentation. The small-scale f ermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zon e) gave similar results. On the other hand, the small-scale well-mixed ferm entation gave the highest biomass yield, but again, surprisingly, the lowes t cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons f or these results are examined in terms of environmental stress associated w ith an ever-increasing glucose limitation in the well-mixed case. On the ot her hand, at the large scale, and to differing degrees in scale-down simula tions, cells periodically encounter regions of relatively higher glucose co ncentration. (C) 2000 John Wiley & Sons, Inc.