Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml−1) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml−1 dropped to ∼10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml−1 probably reflects a loss of cell division capability rather than cell death.

[1]  C. Hewitt,et al.  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. , 2000, Biotechnology and bioengineering.

[2]  C. Hewitt,et al.  Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. , 2000, Journal of microbiological methods.

[3]  S. Enfors,et al.  The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli. , 1994, FEMS microbiology reviews.

[4]  G Larsson,et al.  Rapid sampling, cell inactivation and evaluation of low extracellular glucose concentrations during fed-batch cultivation. , 1996, Journal of biotechnology.

[5]  J. Keller,et al.  Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins inEscherichia coli , 1990, Journal of Industrial Microbiology.

[6]  P. Postma,et al.  Global regulatory networks and signal transduction pathways. , 1999 .

[7]  M. Boaretti,et al.  Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state. , 2003, Environmental microbiology.

[8]  C. Hewitt,et al.  Physiological responses to mixing in large scale bioreactors. , 2001, Journal of biotechnology.

[9]  R. A. Badley,et al.  Viability assessment of bacteria in mixed populations using flow cytometry * , 1995 .

[10]  N. Staten,et al.  Enhanced heterologous gene expression in novel rpoH mutants of Escherichia coli , 1992, Applied and environmental microbiology.

[11]  Howard M. Shapiro,et al.  Practical Flow Cytometry , 1985 .

[12]  S. Enfors,et al.  Modeling of high cell density fed batch cultivation. , 1994, FEMS microbiology reviews.

[13]  W. Goss,et al.  MECHANISM OF ACTION OF NALIDIXIC ACID ON ESCHERICHIA COLI , 1964, Journal of bacteriology.

[14]  C. Hewitt,et al.  Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures. , 1999, Biotechnology and bioengineering.

[15]  E K Gunderson,et al.  ADAPTATION TO EXTREME ENVIRONMENTS: PREDICTION OF PERFORMANCE , 1966 .

[16]  R. R. Colwell,et al.  Viable but nonculturable bacteria: a survival strategy , 2000, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy.

[17]  Christopher J Hewitt,et al.  The application of multi-parameter flow cytometry to monitor individual microbial cell physiological state. , 2004, Advances in biochemical engineering/biotechnology.

[18]  S. Enfors,et al.  Cell Segregation and Lysis Have Profound Effects on the Growth of Escherichia coli in High Cell Density Fed Batch Cultures , 1996, Biotechnology progress.