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Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology
Author(s) -
Sureshkumar G. K.,
Mutharasan R.
Publication year - 1993
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260410116
Subject(s) - intracellular ph , yeast , ph indicator , ionophore , chemistry , intracellular , fluorescence , biochemistry , saccharomyces cerevisiae , bioreactor , nicotinamide adenine dinucleotide , biophysics , chromatography , nad+ kinase , membrane , biology , enzyme , organic chemistry , physics , quantum mechanics
A method has been developed to continuously measure the intracellular pH (pH i ) of cells cultivated in a bioreactor in an on‐line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pH i indicator 9‐aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pH i from measured 9AA‐fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pH i (7 μM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA‐fluorescence. The pH i change in yeast cell 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 pH i , indicator (which is however unsuitable for on‐line applications in a bioreactor) bis‐carboxyethyl carboxy fluorescein (BCECF). The pH i of yeast under substrate starved conditions was 6.4 units. The responses of pH i of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pH i 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, pH i increased by 0.1 unit following a 500‐ppm glucose pulse addition. Comparison of pH i with other indicators of cellular metabolic state suggests that pH i is a cellular metabolic state indicator. © 1993 John Wiley & Sons, Inc.