Effect Of External Ph On The Growth Of Saccharomyces Cerevisiae

Effect o External p H on the Growth o f f S accharomyces cerevisiae Fermenting Maltose in Batch and Continuous Culture
INTRODUCTION
Ma!tose utilization by S. cerevisiae is mediated by an inducible mechanism'.* which is subject to repression and inactivation by g l ~ c o s e Recently it has been reported that the active maltose .~ transport in resting yeasts is not ATP dependent, and is coupledto the electrochemical gradient of protons across the yeast plasma membrane; a stoichiometry of one proton per mole of maltose cotransported has been m e a ~ u r e dThis implies that the external pH should have a great influ.~ ence on maltose-limited growth of S. cerevisiae. We have studied the effect of external proton concentration on exponential growth of this yeast on maltose, in batch andcontinuous cultures.

MATERIAL AND METHODS
Organism and Culture Media
A respiration-deficient mutant of S. cerevisiae 3507. from the Gulbenkian Institute of Science collection (Oeiras, Portugal), was used. The culture medium, with minerals, vitamins, and 0.4% (w/v) maltose (or glucose or galactose, where indicated) was described earlier.' The desired pH was adjusted with K-citrate-phosphate forpH values from 3.5 to 6.0 and with K-phosphates for pH values from 6.0 to 7.0. Cultures were maintained in maltose medium plus 2% agar.

Calculation o Kinetics and Energetics Parameters of Growth in Batch Cultures f
The specific growth rate, p , was calculated as the slope of the regression line to the natural logarithms of the optical density of the culture at 640 nm versus time. The yieldfactor for maltose. Y . was calculated measuring biomass produced and maltose consumed at certain intervals during exponential growth. Biomass produced depended linearly on maltose consumed over these intervals, the slope of the regression line providing an estimate of Y. Biomass, measured as optical density at 640 nm. was related to dry weight by a calibration curve. Maltose concentration wasdetermined by the phenol method' in culture filtrates through Millipore filters (25 mm RAWPO 2500). The specific rate of maltose consumption, q , was calculated from the relation
q = p/Y

(1)

Continuous Culture
A chemostat. LKB 1601 Ultroferm from Biotec (Stockholm, Sweden). with a working volume of 3 L was used. The culture medium was the same as used for batch experiments. At each steady state,the specific consumption rate of maltose was calculated from the relation
q = p / Y =f(S,
-

S)/Vx

(2)

wheref is the flux through the system, S is the maltose concentration in fresh medium. S is the , maltose concentration in the fermenter. V is the volume of culture in the fermentor, and x is the population density. Maltose and biomass were determined as in batch experiments. Themaximum yield factor for maltose, Pa', the maintenance coefficient, m , were oband tained from the Pin equation':
q =m

+D/Pax

(3)

Biotechnology and Bioengineering, Vol. XXIV, Pp. 2725-2729 (1982) CCC 0006-3592/82/122725-05$01 .SO 0 1982 John Wiley & Sons, Inc.

2726

BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIV (1982)

I i

0

0

\

\

-

PH
Fig. 1. Effect of external pH onthe specific growth rate, p , in ( c )mineral medium plus glucose, ( 0 )maltose, and (A) galactose in batch culture.

RESULTS
Effect o External p H in Batch Cultures f

Increases in external pH, over the range tested, decreased the specific growth rate on maltose but not on glucose or galactose (Fig. 1).The yeast was unable to grow on 11.5mM maltose at pH 7. The decrease of p was due todiminution of the specific consumption rate of maltose, q , while the yield factor for the sugar remained constant at all the pH values tested (Table I). The relation between the external proton concentration and the specific rate of maltose consumption was hyperbolic. From a double reciprocal plot, an apparent affinity constant for protons of the uptake system could be calculated, and a value of 2.51...