Influence of aeration on cytoplasmic pH of yeast in an NMR airlift bioreactor.

Abstract

Nuclear magnetic resonance spectroscopy has been increasingly pursued as a tool for noninvasive, real-time studies of metabolic processes of cell suspension in bioreactors. One acute challenge in NMR bioreactor design has been supplying enough oxygen for cell respiration in a suspension that contains sufficient cells for NMR signal detection. The use of cytoplasmic pH as an intracellular marker of adequate oxygenation was evaluated from 31P NMR spectra of the yeast Saccharomyces cerevisiae at several cell densities, ranging from low (0.9% (v/v)) to very high (45% (v/v)) cell densities, in an airlift bioreactor. 31P NMR spectra were obtained for derepressed yeast cells prior to, and during, glycolysis under nongrowth conditions. During endogenous respiration, pHcyt can be used as an intracellular marker for aeration for cell densities up to 18% (v/v) based on two criteria: a value of pHcyt at least 0.2 pH units higher under aerobic than anaerobic conditions and an absolute pHcyt value of 7.1-7.2. These results were more conservative than values of the maximum cell density obtained from calculations using kLa and respiration rate estimates and highlight the utility of intracellular measurements in conjunction with engineering design calculations. During glycolysis, pHcyt values were similar under aerobic and anaerobic conditions and hence pHcyt cannot be used as a marker under these conditions. Carbon dioxide in the influent gas was observed to aid cells in maintaining physiological pHcyt at high cell densities.