Premium
Use of multi‐staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed‐batch cultures
Author(s) -
Hewitt Christopher J.,
NebeVon Caron Gerhard,
Nienow Alvin W.,
McFarlane Caroline M.
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990620)63:6<705::aid-bit8>3.0.co;2-m
Subject(s) - fermentation , flow cytometry , escherichia coli , viable but nonculturable , chromatography , staining , chemistry , biology , cell growth , fed batch culture , biochemistry , cell culture , bacteria , microbiology and biotechnology , genetics , gene
High cell density fed‐batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h −1 , to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD 500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi‐staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed‐batch process is due to cell death and not due to the formation of a “viable but nonculturable state” as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on‐line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 705–711, 1999.