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 coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell 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 the cell size did not change, results confirmed by SEM and TEM. There were 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 latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles.
Hewitt
C J
CJ
Centre for Bioprocess Engineering, School of Chemical Engineering, The University of Birmingham, Edgbaston, B15 2TT, United Kingdom. c.j.hewitt@bham.ac.uk
Boon
L A
LA
McFarlane
C M
CM
Nienow
A W
AW
D019149:Bioreactors; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology*; D005285:Fermentation; D005434:Flow Cytometry / Q000379:methods*; D008566:Membranes / Q000502:physiology; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D011419:Propidium / Q000378:metabolism; D020112:Rhodamine 123 / Q000378:metabolism; D013997:Time Factors
D020112:Rhodamine 123; D011419:Propidium