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The use of multiparameter flow cytometry to examine the state of Escherichia coli during a bioprocess

Shitu, Jennifer Olajumoke; (2005) The use of multiparameter flow cytometry to examine the state of Escherichia coli during a bioprocess. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Traditional monitoring methods of cellular status, such as reproductive viability studies and optical density measurements tend to be time consuming and unable to give any insight into the physiological state of the cell beyond culturability. Multiparameter flow cytometry, a powerful investigative tool that allows the rapid, optical, quantitative measurement of cellular physiology via intrinsic and evoked optical signals from single cell or cell-sized particles in a moving fluid stream, was used as an alternative to provide valuable insights into the effects of bioprocesses on E. coli. Batch fermentation processes and batch and fed batch biocatalytic processes were investigated for this thesis. Multiparameter flow cytometry was used in the characterisation of batch fermentations of E. coli IL-13 during the production of the recombinant human interleukin 13, in conjunction with fermentation profiling by traditional means and recombinant protein characterisation. It was established, via flow cytometric analysis of cell samples stained with a combination of three fluorescent stains, propidium iodide, PI; Bis- (1,3-dibutylbarbituric acid) trimethine oxonol, BOX, and Syto Red 63, SR; that induction with IPTG during the exponential phase, with controlled dissolved oxygen (above 30%) and pH (7 0.2) were the optimum conditions for this fermentation. The changing physiological and metabolic state of the whole cell biocatalyst E. coli TOP 10 pQR239 during the Baeyer-Villiger monooxygenase mediated stereo-selective oxidation of bicyclo (3.2.0.) hept-2-en-6-one to yield (-)-l(S) 5(R)-2-oxabicyclo (3.3.0) oct-6-en-3-one and (-) 1 (R) 5(S)-3-oxabicyclo (3.3.0) oct-6-en-2-one was investigated in batch and fed-batch biocatalytic processes as well as in shake flask bioconversions. The effects of bioconversion media, storage temperature, reaction pH, substrate and product concentration and the influence of the co-substrate glycerol on cell quality and enzyme activity were investigated. Whole cell biocatalyst quality was found to be affected by all of the above mentioned investigated parameters and the presence of the co-substrate was established to be essential for optimum enzyme activity. Multiparameter flow cytometry was successfully applied to an industrially significant fermentation and biocatalytic process and facilitated the rapid analysis of cell quality in both instances to monitor the changes to cell cytoplasmic membrane integrity and polarisation, cell respiration capacity, the production of a recombinant therapeutic protein and a biocatalytic enzyme; thus establishing its feasibility as a tool for process definition and optimisation, during both the research and developmental stages of a bioprocess.

Type: Thesis (Doctoral)
Title: The use of multiparameter flow cytometry to examine the state of Escherichia coli during a bioprocess
Identifier: PQ ETD:602610
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/1446685
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