THE USE OF FLOW-CYTOMETRY TO STUDY THE IMPACT OF FLUID MECHANICAL-STRESS ON ESCHERICHIA-COLI W3110 DURING CONTINUOUS CULTIVATION IN AN AGITATED BIOREACTOR

Continuous culture fermentations of Escherichia coil W3110 have been c arried out at controlled dissolved oxygen levels of 40% and 10% of sat uration. Satisfactory and reproducible results were obtained. Agitatio n speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been us ed as well as an aeration rate of 3 vvm at 400 rpm. The upper levels o f these variables represent much higher agitation and aeration intensi ties than those normally used in practical fermentations. The fermenta tions were monitored by mass spectrometry and optical density, and cel l samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also th e cell size did not change, results confirmed by SEM and TEM. There we re no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latte r conditions, after 7 h, the outer polysaccharide layer on the cell wa s stripped away. It is concluded that any changes in biological perfor mance of this E. coli cell line due to variations in agitation or aera tion intensity or scale of operation cannot be attributed to fluid dyn amic stresses associated with the turbulence generated by impellers or with bursting bubbles. (C) 1998 John Wiley & Sons, Inc. Biotechnol Bi oeng 59: 612-620, 1998.