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

The intracellular pH (pH(i) of alpha-omega-dicarylic acid producing Candida tropicalis was determined by a fluorescence technique using the pH-sensitive fluorescent probe 5(6)-carboxyfluorescein diacetate. Fermentations with n-tridecane substrate to produce alpha,omega-tridecanedioic acid were carried out in a 5-l bioreactor in which growth and production were separated. During the growth phase, the measured pH(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(i). Cytochrome P450 monooxygenase (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(i). A much higher level of P450 activity was observed at pH(i) of 6.55+/-0.15 for all the fermentations, with maximum productivity (1.919 g/l/h) occurring when using an optimal pH-control strategy. However, P450 activity, tridecanedioic acid productivity, and pH(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(i), the metabolic activity of C. tropicalis is sensitive to the variations in pH(i). The measured pH(i) varied from 6.1 to 6.7 during the production phase for all the fermentations. Thus, both tridecanedioic acid productivity and P450 activity are correlated with pH(i) or pH gradients across the cell membrane.