Intracellular pH and metabolic activity of long-chain dicar☐ylic acid-producing yeast Candida tropicalis

Abstract

The intracellular pH (pH i of α-ω-dicar☐ylic acid producing Candida tropicalis was determined by a fluorescence technique using the pH-sensitive fluorescent probe 5(6)-car☐yfluorescein diacetate. Fermentations with n-tridecane substrate to produce α,ω-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, CO 2 production, and O 2 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.