Intracellular pH and metabolic activity of long-chain dicarboxylic acid-producing yeast Candida tropicalis

The intracellular pH (pH sub(i)) of a,o-dicarboxylic acid producing Candida tropicalis was determined by a fluorescence technique using the pH-sensitive fluorescent probe 5(6)-carboxyfluores-cein diacetate. Fermentations with n-tridecane substrate to produce a,o-tridecanedioic acid were carried out in a 5-l bioreactor in which growth and production were separated. During the growth phase, the measured pH sub(i) values were varied from 5.65 to 6.15 in all the experiments performed under different constant pH-operating conditions. The specific rates of growth, sucrose consumption, CO2 production, and O2 consumption were correlated with pH sub(i). Cytochrome P450 monooxy-geuase (P450), which catalyzes n-alkane hydroxylation, was only slightly expressed during the growth phase. During the first 6 h of the production phase, P450 activity was induced rapidly accompanying higher pH sub(i). A much higher level of P450 activity was observed at pH sub(i) of 6.55=+-0.15 for all the fermentations, with maximum productivity (1.919 g/l/h) occurring when using all optimal pH-control strategy. However, P450 activity, tridecanedioic acid productivity, and pH sub(i) decreased progressively during the latter part of the production phase, as a consequence of 'the metabolic activity changes of the cells. Even though culture pH has only a slight influence on pH sub(i), the metabolic activity of C. tropicalis is sensitive to the variations in pH sub(i). The measured pH sub(i) varied from 6.1 to 6.7 during the production phase for all the fermentations. Thus, both tridecalredioic acid productivity and P450 activity are correlated with pH sub(i) or pH gradients across the cell membrane.