Endogenously produced CO2 induces stationary phase in tetrahymena cultures

Media concentration of total soluble CO₂ increases with culture age of Tetrahymena pyriformis. CO₂ is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO₂ produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO₂. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO₂/HCO. In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO₂, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO₂ concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO₂ in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.