Intracellular pH based controlled cultivation of yeast cells. I. Measurement methodology

A method has been developed to continuously measure the intracellular pH (pHi) of cells cultivated in a bioreactor in an on-line fashion over extended time periods. The method is attractive in its simplicity and involves the use of a fluorescent phi indicator 9-aminoacridine (9AA) which is a weak ba...

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Bibliographic Details
Published in:Biotechnology and bioengineering 1993, Vol.41 (1), p.118-128
Main Authors: Sureshkumar, G.K, Mutharasan, R
Format: Article
Language:English
Subjects:
9-aminoacridine
acridines
Biological and medical sciences
bioreactor
bioreactors
Biotechnology
cytoplasm
fluorescence
fluorescent dyes
Fundamental and applied biological sciences. Psychology
intracellular pH
measurement
Methods. Procedures. Technologies
Microbial engineering. Fermentation and microbial culture technology
monitoring
on-line measurement
Saccharomyces cerevisiae
yeast
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Summary:A method has been developed to continuously measure the intracellular pH (pHi) of cells cultivated in a bioreactor in an on-line fashion over extended time periods. The method is attractive in its simplicity and involves the use of a fluorescent phi indicator 9-aminoacridine (9AA) which is a weak base. An expression has been derived to calculate changes in pHi from measured 9AA-fluorescence changes. The indicator 9AA was found to be nontoxic to yeast cells at concentrations used to measure phi (7 micromolar). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA-fluorescence. The pHi change in yeast cells following the addition of a proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well-established pHi indicator (which is however unsuitable for on-line applications in a bioreactor) bis-carboxyethyl carboxy fluorescein (BCECF). The pHi of yeast under substrate starved conditions was 6.4 units. The responses of pHi of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pHi increased by 0.12 unit following a 100-ppm glucose pulse addition and by 0.25 unit following a 300-ppm ethanol pulse addition. Under anaerobic condition, pHi increased by 0.1 unit following a 500-ppm glucose pulse addition. Comparison of pH, with other indicators of cellular metabolic state suggests that pHi is a cellular metabolic state indicator.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.260410116