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Open Access A Study of the Physiological Response of Pichia pastoris Growing in a Continuous Culture to an Induced Stress Situation

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During our work, flow cytometry was used for a study of the influence of cultivation conditions on a physiological state, mainly on the viability and vitality of the recombinant Pichia pastoris strain producing β-galactosidase. The fluorescent dyes propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol were used for the determination of the structural integrity and the membrane potential of Pichia pastoris cells, respectively. First the staining procedures were optimised and adapted for the Pichia pastoris yeast cells and later applied in a lab-scale continuous cultivation. Stained samples were analysed with the flow cytometer PAS III Partec or the epifluorescence microscope Nikon Eclipse E400. The following conditions were found to be optimum for the staining of Pichia pastoris cells: 57 μg/ml propidium iodide in the sample, 5 min incubation time; 2 μg/ml bis-(1,3-dibutylbarburic acid) trimethine oxonol in the sample, 20 min incubation time. The optimised staining methods were employed in a study of stress-induced physiological response to change of substrate from glycerol to methanol in a glycerol steady state growing culture of Pichia pastoris yeast. During the first 5 h of the transitional state an accumulation of methanol in the culture broth was accompanied by a decreasing concentration of biomass and an increasing amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol. After the adaptation phase the amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol reached steady levels of 2% and 5%, respectively.

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Keywords: CONTINUOUS CULTURE; FLOW CYTOMETRY; PHYSIOLOGY; PICHIA PASTORIS

Document Type: Research Article

Publication date: October 1, 2005

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  • International Journal for Chemistry and Official Membership Journal of the Swiss Chemical Society (SCS) and its Divisions

    CHIMIA, a scientific journal for chemistry in the broadest sense, is published 10 times a year and covers the interests of a wide and diverse readership. Contributions from all fields of chemistry and related areas are considered for publication in the form of Review Articles and Notes. A characteristic feature of CHIMIA are the thematic issues, each devoted to an area of great current significance.

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