Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology.
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 microM). 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.
Sureshkumar
G K
GK
Department of Chemical Engineering, Drexel University, Philadelphia, Pennsylvania 19104, USA.
Mutharasan
R
R