Archives of
Microbiology
Arch. Microbiol. 113, 303-307 (1977)
9 by Springer-Verlag 1977
Isotherrnic Variation of the Specific Growth Rate of Saccharomyces cerevisiae in Batch Culture * J. M A R T I N E Z - P E I N A D O * * and N. VAN U D E N Laboratory of Microbiology, Gulbenkian Institute of Science, Oeiras, Portugal
Abstract. The specific growth rate (#) of a respirationdeficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25 ~ C) varied from experiment to experiment over a wide range ( 0 . 1 0 - 0 . 2 4 h -1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of #. The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO2. The yield decreased with # implying higher maintenance requirements in batch culture (4.7 mmoles g-1 h - i ) than in continuous culture (0.8 mmoles g-1 h - l ) . It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO2 production, and of the yield appeared to be consequences rather than causes of the variation of #. The ultimate causes of the variation of # remained unidentified. Saccharomyces cerevisiae - Specific growth rate - Growth control - Glucose transfer Glucose-6-phosphate - Maintenance requirements. Key words."
reactor is usually defined as #-
dx dt
1 x
(1)
where x is the instantaneous population density. The specific growth rate is a function of the concentration of the nutrients, having a maximum value (#max) when all nutrients have saturating concentrations (Monod, 1942). The maximum specific growth rate is generally thought to be a constant for a given strain in a growth medium of given composition if certain environmental conditions are kept constant, such as the temperature, pH, agitation, aeration, etc. However, Stanley (1964) found that #max of the yeast Candida utilis growing in batch culture under controlled conditions varied from experiment to experiment for unidentified reasons. We have observed a similar behavior in a respiratory mutant of the yeast Saccharomyces cerevisiae. In the present paper we report on the statistics of this variation and on its correlation with other parameters of growth and metabolism. MATERIAL AND METHODS Organism and Culture Media. A respiration deficient mutant was used, prepared earlier (van Uden, 1967b) from a strain of Saccharom y c e s cerevisiae which is maintained in our collection under IGC
3507.
The culture mediumfor growth experimentsCMGV medium") contained minerals and vitamins, in addition to 2 % glucose (w/v). The composition and preparation of this medium was described earlier (van Uden, 1967b). Cultures were maintained on MGV medium solidifiedwith 2 % agar. The specific growth rate of a microbial population growing exponentially in liquid medium in a stirred * Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra, Spain ** P r e s e n t address: Department of Biology, Faculty of Sciences, University of Extremadura, Badajoz, Spain
Calculation of the Specific Growth Rate. Cultures were grown in
250 ml Erlenmeyerflasks containing 100 ml of MGV medium. The flasks were maintained at 25~C in a water bath and their contents agitated by magnetic stirring. No forced aeration was necessary since the yeast strain was respiration deficient. Samples were taken at regular intervals during the exponential phase and their optical density (OD) measured in a Bausch and Lomb, Spectronic 20, spectrophotometer at 640 nm.
304
Z uJ
Arch. Microbiol., Vol, 113 (1977)
0.5
0,200 "
O
~.
~
O"
0
0 0 0 _ o- ~ oOo~----~o~
[a_ OA
/,
0.2
-~ 20
~, 0,100
t
0.060
0.120
1
0.180
P (h-l)
5
0.240 2
I
1; TIME (h)
~"~
1~
I
0.050
25 3
I
0.100 p (h-%
I
I
0.150
0.200
Fig. 1. Distribution of the specific growth rates of Saccharomyces cerev&iae in 40 exponential batch cultures at 25 ~ C in mineral medium with glucose and vitamins. Solid line: frequency polygon. Dashed line: normal curve with the same mean and standard deviation Fig. 2. Variation with time of the specific growth rate of Saccharomyces cerevisiae in an exponential batch culture at 25 ~C in mineral medium with glucose and vitamins Fig. 3. Variation of the yield with respect to glucose of Saccharomyces cerevisiae in 9 batch cultures at 25 ~C in mineral medium with glucose and vitamins. The curve fitted to the experimental points was calculated according to Equation (2) using estimates of Y~l~"2and k,, obtained from Figure 4
From least square fits to the natural logarithms of the OD values, #,..~ was calculated using the following relation: In OD~ = In ODo + #m~ t.
(2)
Table 1. Variation of the specific growth rate of Saccharomyces cerevisiae in 80 exponential batch cultures at 25~ in mineral medium with glucose and vitamins
Manometry. Manometric measurements of CO2 evolution by grow-
Type of inoculum
ing cultures were made by the direct Warburg method (Umbreit et al., 1964). The cultures were grown in 125 ml Warburg flasks containing 50 ml of MGV medium. Control experiments with flasks containing KOH showed that no gases other than COz were consumed or produced in measurable quantities.
Number of Mean experiments _+ S.E. a
Variation coefficient + S.E. a
Cells from solid medium
40
0.17 h -I • 0.006
22.4 _+ 2.6
Ceils from liquid medium
40
0.16 h -1 • 0.006
22.9 + 2.7
Analytical Procedures. Samples of cultures were filtered through Millipore filters (25 mm RAWPO 2500). Glucose and ethanol were estimated in the filtrates by the use of kits of Boehringer Mannheim GmbH, Germany, based on the glucose oxidase and alcohol dehydrogenase method, respectively (Bergmeyer, 1962). Yields with respect to glucose (yg~,o) were calculated by dividing biomass produced b y glucose consumed. The biomass was estimated by determining OD640 and reading the corresponding dry weight value on a calibration curve.
RESULTS
Effects of the Nature of the Inoeulum on the Variation of the Specific Growth Rate The specific growth rates obtained in 80 batch cultures that had been inoculated from solid or liquid media varied over a wide range (0.10-0.24 h-l). The variability of/~, as expressed by the variation coefficient (Table 1), was the same in the cultures inoculated from either medium and in both cases the distribution of # was normal (P < 0.01, Kolmogorov-Smirnov test: Sokal and Rohlf,/966). Figure 1 depicts the frequency polygen of # obtained in cultures inoculated from solid medium and the corresponding normal curve. The means of the two sets of specific growth rates were not significantly different at the 1 ~ level and
"
Standard error
were just so within the limit of the 5 ~ level. So, additional experiments were needed to decide whether the nature of the inoculum has a significant effect on the value of/x. In a second series of experiments the inoculum was standardized as follows: 50 ml of liquid medium in an 150-ml Erlenmeyer flask were inoculated from a 24 h slant and incubated overnight at 25~ with shaking. The grown culture was transferred to fresh liquid medium in sufficient amount for an initial population density corresponding to an OD640 of 0.1 and the growth experiment was started. The values of # obtained in 11 batch cultures of this type showed a decreased variability but had still a variation coefficient of 15.7 ~.
Variation of the Specific Growth Rate of the Same Culture with Age To estimate the variability of # in the course of the same growth experiment, growth of a culture inocu-
J. Martinez-Peinado and N. van Uden: Growth Rate Variation in Saccharomyces eerevisiae
lated from solid medium was measured during 30 h and the specific growth rates calculated for a number of time intervals during exponential growth. There was a significant negative correlation between the values of the specific growth rate and the age of the culture, in addition to random variation conceivably due to experimental error (Fig. 2). However, the variation coefficient was only 9.6 __+ 1.53 ~o which is significantly (P < 0.01) lower than the variation coefficients obtained for the variation of # among different growth experiments (Table 1). Thus, neither the age of the culture, the history and the state of the inoculum nor experimental error did account wholy for the variation of the specific growth rate.
Relation between the Growth Yield and the Variation of the Specific Growth Rate Figure 3 depicts the experimental estimates of the yield with respect to glucose (yg~ue)plotted against #. Inspection of the data suggested a decrease of yglue with decreasing values of g. Though the correlation between the two variables was not statistically significant within the 5 ~ limit, its possible biological significance was further explored. In chemostat cultures of heterotrophic microorganisms the yield with respect to the carbon source varies with #, due to maintenance requirements. If no other sources of yield variation are simultaneously present, this variation may be represented by a hyperbolic function (van Uden, 1971): Yglue
=
~x glue
#
-
ymax k m + #
(2)
glue
where k,,, the so-called maintenance coefficient (Pitt, 1965, 1975), represents here the specific transfer rate of glucose for maintenance, while Yg'~u~,the maximum or the true yield, represents the limit of Yglue for high values of g. Taking reciprocals on both siges of (2) and multiplying by #, lead to the linear relation originally proposed by Pirt (1965): kglue = km + ~
1
#.
(3)
Values of kg~ue, the specific transfer rate of glucose, may be calculated using (van Uden, 1967a): kgluc
--
# yg,uc
(4)
A plot of the kgluo values against p yielded a linear relation (Fig. 4). From this plot and using Equation (3), the following estimates were obtained: k~ 4.7 mmoles g Zh -1 and Yg~ 16.9gmol -~. Introducing these
305
r
20 o
10
~ i
0.050
~176 l
0.100
I
0.15o
I
0.200
ju (h -~) Fig. 4. Variation of the specific transfer rate of glucose in Saccharoin mineral medium with glucose and vitamins
myces cerevisiae in 9 batch cultures at 25~
estimates in Equation (2), a hyperbolic plot could be fitted to the experimental yield data (Fig. 3). In as far as this treatment of batch culture data is legitimate (see "Discussion"), it may be concluded that the variation of the yield was a consequence rather than a cause of the variation of the specific growth rate.
Relation between the Specific Transfer Rate of Glucose and the Variation of the Specific Growth Rate The correlation between kglue and p (Fig. 4) was positive and significant with a Pearson coefficient of 0.803. To obtain some information on whether external or intracellular factors determined the correlated variability of # and kglue, the following experiments were carried out. Two exponential cultures at different rates were harvested by centrifugation. After resuspension of the cells in fresh medium and a short lag, exponential growth was resumed with the same # value each culture had displayed before the washing. This showed that the rate differences were not due to effects of metabolites excreted into the growth medium. In another experiment, three cultures with different specific growth rates were harvested by centrifugation. One half of the cells of each culture was resuspended in fresh MGV medium, the other half in fresh MGV medium that lacked a source of nitrogen. While the first resumed growth, the latter (resting cells) fermented glucose without growing. CO2 production was estimated manometrically. The rates of CO2 production by the resting cell preparations were independent of the previous # values and had the same value in all three (10.4mmolesg -1 h-l). The specific rates of growth and CO2 production in the growing preparations were similar to those of the original cultures. These results seemed to indicate that intracellular factors control the specific rates of growth and of glucose transfer and that this control is lost in resting cells.
306
Arch. Microbiol., Vol. 113 (1977)
A
"7
10
~c i c~
E E
"~o..
5
O u
9 . . . .
I 0.100
j 0.150
,% ~ o - i q~
- -o--
0.200
,u (h -I)
Fig. 5. Variation of the specific rate of non-fermentative CO2 production by Saccharomyces cerevisiae in 12 batch cultures at 25~ in mineral medium with glucose and vitamins
Relation between the Production of Non-Fermentative C02 and the Variation of the Specific Growth Rate Another difference between the resting and growing cells was the production of non-fermentative COz. In resting cells the molar amounts of ethanol and CO2 produced were identical, showing that all CO2 resulted from alcoholic fermentation. In growing cells the production of CO2 exceeded the production of ethanol. The production of non-fermentative CO2 depended on the specific growth rate (Fig. 5). At high values of/~ (0.23 h-1 _ 0.19 h-a), the specific rate of non-fermentative CO2 production was low and nearly constant (0.4 mmoles g-a h-a). At lower values of # the correlation with the specific rate of non-fermentative CO2 production was negative, the latter increasing to a value of 5.1 mmoles g-1 h-a at the lowest experimental g value (0.102 h-a), i.e. 34% of the total specific rate of CO2 production.
DISCUSSION The finding that the distribution of the specific growth rate in our strain of Saccharomyces cerevisiae was normal suggests that many factors, additive and independent in occurrence (Sokal and Rohlf, 1966) determine its variation. Neither the nature of these factors nor their primary targets have been identified. Bortels (1951) and his co-workers have reported on correlations between the rates of many microbial activities, including growth, and meteorological conditions. We became aware of his work after the conclusion of our experiments which had not been planned in a way that permitted to search a posteriori for such correlations. The influence of the history of the inoculum on the subsequent specific growth rate, as well as the lack of effect of washing the cells of exponentially
growing cultures on their specific growth rates, suggest that a number of possible physiological states may be established, which predetermine the specific growth rate during an extended period of time. In other words, each of these pysiological states appears to have some build-in inertia. This inertia is conceivably an expression of resistance to change by control mechanisms calibrated to maintain a given # value, imposed earlier on the cell population by unidentified factors. This concept implies that the observed variations of the yield (Ygluo) and of the specific rate of glucose (kgluc) were consequences rather than causes of the variation of the specific growth rate. Formal treatment of the variation of yg],c led to an estimate of kin, the maintenance coefficient, which was about six times higher than an estimate of k,, obtained earlier (van Uden, 1971) for the same strain in a glucose-limited chemostat culture in the same basal medium: 4.7 mmoles g-1 h-~ as compared with 0.8 mmoles g-a h-t. Thus, if the treatment is indeed applicable the conclusions would be, firstly, that maintenance requirements are substantially higher in batch cultures than in carbon-limited chemostat cultures and, secondly, that in batch cultures the maintenance requirements may be high enough to affect, measurably, the values of the yield. That this may well be the case was made more likely by the fact that y ~ x calculated for the set of batch cultures was very similar with the estimate obtained earlier (van Uden, 1971) for the same strain in the chemostat: 16.9 g mol -a as compared with 15.3 g mol-t. Similar relations were encountered (de Vries et al., 1970; Stouthamer and Bettenhausen, 1973) with a strain of Lactobacillus casei: the maintenance coefficient calculated from batch culture data was about three times higher than the estimate obtained in chemostat cultures. The notion that the variation of the specific growth rate determined the variation of the specific transfer rate of glucose, rather than the other way round, received support from the finding that control of kg]uc was lost when growing cells were forcibly changed into resting cells by eliminating nitrogen nutrient from the medium. Feed-back control of glucose transport in Saccharomyces cerevisiae is thought to be effected by an intracellular metabolite (Sols et al., 1971 ; Serrano and de la Fuente, 1974) which has tentatively been identified as glucose-6-phosphate (Azam and Kotyk, 1969; Becker and Betz, 1972). Our finding of the negative correlation between the specific growth rate and the specific rate of production of non-fermentative COz points to glucose-6-phosphate as a regulator. Increases in the pool size of this compound might simultaneously increase non-fermentative CO2 production through stimulation of the hexose monophosphate pathway
J. Martinez-Peinado and N. van Uden: Growth Rate Variation in Saccharornyces cerevisiae
and depress the transfer of glucose into the cell through feed-back inhibition of its transport. In yeasts, as in other microorganisms, the R N A content of the cells increase with increasing specific growth rate. Based on R N A measurements in S. cerevisiae by McMurrough and Rose (1967), we calculated an approximate ribose content of 220 Ixmoles per gram of dry weight of cells growing at a specific rate of 0.24 h -s, the highest/~ value we obtained. This corresponds to a specific rate of ribose production of about 53 gmoles g-~ h -~. The lowest specific rate of non-fermentative CO2 production we obtained (400 gmoles g-1 h-S) exceeds this maximum need by about one order of magnitude. Thus the observed negative correlation between the specific rates of growth and of non-fermentative CO2 production, does not conflict which the need for increased ribose production at higher specific growth rates. A possible explanation of the negative correlation may reside in the catabolism of carbohydrate reserves which occurs shortly before budding (yon Meyenburg, 1969; Kfienzi and Fiechter, 1969), the extent of which was negatively correlated with the specific growth rate. Though at least part of the reserves were catabolised by alcoholic fermentation, the data of those authors do not exclude a significant contribution of the hexose monophosphate pathway. Though the observed correlations between the variation of the specific growth rate and other parameters of growth may be of interest in themselves, the main finding consists in the observed isothermic variation of the specific growth rate under conditions of nutrient saturation. We conclude that #maxas defined by Monod (1942) is not a constant in the strain we studied, as it was not in the strain of Candida utilis by Stanley (1964). It remains to be seen whether this behavior is of general occurrence among microorganisms.
REFERENCES Azam, F., Kotyk, A. : Glucose-6-P as regulator of monosaccharide transport in baker's yeast. FEBS Letters 2, 333-335 (1969)
307
Becker, J. V., Betz, A.: Membrane transport as controlling pace. maker in Saccharomyces carlsbergensis. Biochim. biophys. Acta (Amst.) 274, 584-597 (1972) Bergmeyer, H. U. : Methoden der enzymatischen Analyse. Weinheim: Verlag Chemic 1962 Bortels, H. : Beziehungen zwischen Witterungsablauf, physikalischchemischen Reaktionen, biologischem Geschehen und Sonnenaktivit/it. Naturwissenschaften 38, 165 - 176 (1951) K~ienzi, M. T., Fiechter, A. : Changes in carbohydrate composition and trehatose-activity during the budding cycle of Saecharomyces cerevisiae. Arch. Mikrobiol. 64, 396-407 (1969) McMurrough, I., Rose, A. H. : Effect of growth rate and substrate limitation on the composition and structure of the cell wall of Saccharomyces cerevisiae. Biochem. J. 105, 189-203 (1967) von Meyenburg, H. K. : Energetics of the budding cycle of Saccharomyees eerevisiae during glucose limited aerobic growth. Arch. Mikrobiol. 66, 289-303 (1969) Monod, J_ : Recherches sur la croissance des cultures bact6riennes. Paris: Herman 1942 Pitt, S. J. : The maintenance energy of bacteria in growing cultures. Proc. roy. Soc. B 163, 224-231 (1965) Pirt, S. J. : Principles of microbe and cell cultivation. Oxford: Blackwell 1975 Serrano, R., de la Fuente, G. : Regulatory properties of the constitutive hexose transport in Saecharomyces cerevisiae. Molec. Cell. Biochem. 6, 161-171 (1974) Sokal, R. R., Rohlf, F. J. : Biometry. San Francisco: Freeman 1966 Sols, A., Gancedo, C., de la Fuente, G. : Energy yielding metabolism in yeast. In: The yeast, Vol. II (A. H. Rose, J. S. Harrison, eds.), pp. 271-307. London-New York: Academic Press 1971 Stanley, P. E. : The growth of Torulopsis utilis in aerated batch and continuous cultures. Ph.D. Thesis, University of Bristol (1964) Stouthamer, A. H., Bettenhaussen, C. : Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim. biophys. Acta (Amst.) 301, 53-70 (1973) van Uden, N. : Transport limited growth in the chemostat and its competitive inhibition; a theoretical treatment. Arch. Mikrobiol. 58, 145-154 (1967a) van Uden, N. : Transport limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. MikrobioL 58, 155-166 (1967b) van Uden, N. : Kinetics and energetics of yeast growth. In: The yeasts, Vol. II (A. H. Rose, J. S. Harrison, eds.), pp. 75-118. London-New York: Academic Press 1971 de Vries, W., Kapteijn, W. M. C., van der Beck, E. G., Stouthamer, A . H . : Molar growth yields and fermentation balances of Lactobacillus easei L3 in batch cultures and in continuous cultures. J. gen. Microbiol. 63, 333-345 (1970) Umbreit, W. W., Burris, R. H., Stauffer, J. F. : Manometric techniques, 4th ed. Minneapolis, Minnesota: Burgess 1964
Received January 21, 1977
Microbiology
Arch. Microbiol. 113, 303-307 (1977)
9 by Springer-Verlag 1977
Isotherrnic Variation of the Specific Growth Rate of Saccharomyces cerevisiae in Batch Culture * J. M A R T I N E Z - P E I N A D O * * and N. VAN U D E N Laboratory of Microbiology, Gulbenkian Institute of Science, Oeiras, Portugal
Abstract. The specific growth rate (#) of a respirationdeficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25 ~ C) varied from experiment to experiment over a wide range ( 0 . 1 0 - 0 . 2 4 h -1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of #. The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO2. The yield decreased with # implying higher maintenance requirements in batch culture (4.7 mmoles g-1 h - i ) than in continuous culture (0.8 mmoles g-1 h - l ) . It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO2 production, and of the yield appeared to be consequences rather than causes of the variation of #. The ultimate causes of the variation of # remained unidentified. Saccharomyces cerevisiae - Specific growth rate - Growth control - Glucose transfer Glucose-6-phosphate - Maintenance requirements. Key words."
reactor is usually defined as #-
dx dt
1 x
(1)
where x is the instantaneous population density. The specific growth rate is a function of the concentration of the nutrients, having a maximum value (#max) when all nutrients have saturating concentrations (Monod, 1942). The maximum specific growth rate is generally thought to be a constant for a given strain in a growth medium of given composition if certain environmental conditions are kept constant, such as the temperature, pH, agitation, aeration, etc. However, Stanley (1964) found that #max of the yeast Candida utilis growing in batch culture under controlled conditions varied from experiment to experiment for unidentified reasons. We have observed a similar behavior in a respiratory mutant of the yeast Saccharomyces cerevisiae. In the present paper we report on the statistics of this variation and on its correlation with other parameters of growth and metabolism. MATERIAL AND METHODS Organism and Culture Media. A respiration deficient mutant was used, prepared earlier (van Uden, 1967b) from a strain of Saccharom y c e s cerevisiae which is maintained in our collection under IGC
3507.
The culture mediumfor growth experimentsCMGV medium") contained minerals and vitamins, in addition to 2 % glucose (w/v). The composition and preparation of this medium was described earlier (van Uden, 1967b). Cultures were maintained on MGV medium solidifiedwith 2 % agar. The specific growth rate of a microbial population growing exponentially in liquid medium in a stirred * Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra, Spain ** P r e s e n t address: Department of Biology, Faculty of Sciences, University of Extremadura, Badajoz, Spain
Calculation of the Specific Growth Rate. Cultures were grown in
250 ml Erlenmeyerflasks containing 100 ml of MGV medium. The flasks were maintained at 25~C in a water bath and their contents agitated by magnetic stirring. No forced aeration was necessary since the yeast strain was respiration deficient. Samples were taken at regular intervals during the exponential phase and their optical density (OD) measured in a Bausch and Lomb, Spectronic 20, spectrophotometer at 640 nm.
304
Z uJ
Arch. Microbiol., Vol, 113 (1977)
0.5
0,200 "
O
~.
~
O"
0
0 0 0 _ o- ~ oOo~----~o~
[a_ OA
/,
0.2
-~ 20
~, 0,100
t
0.060
0.120
1
0.180
P (h-l)
5
0.240 2
I
1; TIME (h)
~"~
1~
I
0.050
25 3
I
0.100 p (h-%
I
I
0.150
0.200
Fig. 1. Distribution of the specific growth rates of Saccharomyces cerev&iae in 40 exponential batch cultures at 25 ~ C in mineral medium with glucose and vitamins. Solid line: frequency polygon. Dashed line: normal curve with the same mean and standard deviation Fig. 2. Variation with time of the specific growth rate of Saccharomyces cerevisiae in an exponential batch culture at 25 ~C in mineral medium with glucose and vitamins Fig. 3. Variation of the yield with respect to glucose of Saccharomyces cerevisiae in 9 batch cultures at 25 ~C in mineral medium with glucose and vitamins. The curve fitted to the experimental points was calculated according to Equation (2) using estimates of Y~l~"2and k,, obtained from Figure 4
From least square fits to the natural logarithms of the OD values, #,..~ was calculated using the following relation: In OD~ = In ODo + #m~ t.
(2)
Table 1. Variation of the specific growth rate of Saccharomyces cerevisiae in 80 exponential batch cultures at 25~ in mineral medium with glucose and vitamins
Manometry. Manometric measurements of CO2 evolution by grow-
Type of inoculum
ing cultures were made by the direct Warburg method (Umbreit et al., 1964). The cultures were grown in 125 ml Warburg flasks containing 50 ml of MGV medium. Control experiments with flasks containing KOH showed that no gases other than COz were consumed or produced in measurable quantities.
Number of Mean experiments _+ S.E. a
Variation coefficient + S.E. a
Cells from solid medium
40
0.17 h -I • 0.006
22.4 _+ 2.6
Ceils from liquid medium
40
0.16 h -1 • 0.006
22.9 + 2.7
Analytical Procedures. Samples of cultures were filtered through Millipore filters (25 mm RAWPO 2500). Glucose and ethanol were estimated in the filtrates by the use of kits of Boehringer Mannheim GmbH, Germany, based on the glucose oxidase and alcohol dehydrogenase method, respectively (Bergmeyer, 1962). Yields with respect to glucose (yg~,o) were calculated by dividing biomass produced b y glucose consumed. The biomass was estimated by determining OD640 and reading the corresponding dry weight value on a calibration curve.
RESULTS
Effects of the Nature of the Inoeulum on the Variation of the Specific Growth Rate The specific growth rates obtained in 80 batch cultures that had been inoculated from solid or liquid media varied over a wide range (0.10-0.24 h-l). The variability of/~, as expressed by the variation coefficient (Table 1), was the same in the cultures inoculated from either medium and in both cases the distribution of # was normal (P < 0.01, Kolmogorov-Smirnov test: Sokal and Rohlf,/966). Figure 1 depicts the frequency polygen of # obtained in cultures inoculated from solid medium and the corresponding normal curve. The means of the two sets of specific growth rates were not significantly different at the 1 ~ level and
"
Standard error
were just so within the limit of the 5 ~ level. So, additional experiments were needed to decide whether the nature of the inoculum has a significant effect on the value of/x. In a second series of experiments the inoculum was standardized as follows: 50 ml of liquid medium in an 150-ml Erlenmeyer flask were inoculated from a 24 h slant and incubated overnight at 25~ with shaking. The grown culture was transferred to fresh liquid medium in sufficient amount for an initial population density corresponding to an OD640 of 0.1 and the growth experiment was started. The values of # obtained in 11 batch cultures of this type showed a decreased variability but had still a variation coefficient of 15.7 ~.
Variation of the Specific Growth Rate of the Same Culture with Age To estimate the variability of # in the course of the same growth experiment, growth of a culture inocu-
J. Martinez-Peinado and N. van Uden: Growth Rate Variation in Saccharomyces eerevisiae
lated from solid medium was measured during 30 h and the specific growth rates calculated for a number of time intervals during exponential growth. There was a significant negative correlation between the values of the specific growth rate and the age of the culture, in addition to random variation conceivably due to experimental error (Fig. 2). However, the variation coefficient was only 9.6 __+ 1.53 ~o which is significantly (P < 0.01) lower than the variation coefficients obtained for the variation of # among different growth experiments (Table 1). Thus, neither the age of the culture, the history and the state of the inoculum nor experimental error did account wholy for the variation of the specific growth rate.
Relation between the Growth Yield and the Variation of the Specific Growth Rate Figure 3 depicts the experimental estimates of the yield with respect to glucose (yg~ue)plotted against #. Inspection of the data suggested a decrease of yglue with decreasing values of g. Though the correlation between the two variables was not statistically significant within the 5 ~ limit, its possible biological significance was further explored. In chemostat cultures of heterotrophic microorganisms the yield with respect to the carbon source varies with #, due to maintenance requirements. If no other sources of yield variation are simultaneously present, this variation may be represented by a hyperbolic function (van Uden, 1971): Yglue
=
~x glue
#
-
ymax k m + #
(2)
glue
where k,,, the so-called maintenance coefficient (Pitt, 1965, 1975), represents here the specific transfer rate of glucose for maintenance, while Yg'~u~,the maximum or the true yield, represents the limit of Yglue for high values of g. Taking reciprocals on both siges of (2) and multiplying by #, lead to the linear relation originally proposed by Pirt (1965): kglue = km + ~
1
#.
(3)
Values of kg~ue, the specific transfer rate of glucose, may be calculated using (van Uden, 1967a): kgluc
--
# yg,uc
(4)
A plot of the kgluo values against p yielded a linear relation (Fig. 4). From this plot and using Equation (3), the following estimates were obtained: k~ 4.7 mmoles g Zh -1 and Yg~ 16.9gmol -~. Introducing these
305
r
20 o
10
~ i
0.050
~176 l
0.100
I
0.15o
I
0.200
ju (h -~) Fig. 4. Variation of the specific transfer rate of glucose in Saccharoin mineral medium with glucose and vitamins
myces cerevisiae in 9 batch cultures at 25~
estimates in Equation (2), a hyperbolic plot could be fitted to the experimental yield data (Fig. 3). In as far as this treatment of batch culture data is legitimate (see "Discussion"), it may be concluded that the variation of the yield was a consequence rather than a cause of the variation of the specific growth rate.
Relation between the Specific Transfer Rate of Glucose and the Variation of the Specific Growth Rate The correlation between kglue and p (Fig. 4) was positive and significant with a Pearson coefficient of 0.803. To obtain some information on whether external or intracellular factors determined the correlated variability of # and kglue, the following experiments were carried out. Two exponential cultures at different rates were harvested by centrifugation. After resuspension of the cells in fresh medium and a short lag, exponential growth was resumed with the same # value each culture had displayed before the washing. This showed that the rate differences were not due to effects of metabolites excreted into the growth medium. In another experiment, three cultures with different specific growth rates were harvested by centrifugation. One half of the cells of each culture was resuspended in fresh MGV medium, the other half in fresh MGV medium that lacked a source of nitrogen. While the first resumed growth, the latter (resting cells) fermented glucose without growing. CO2 production was estimated manometrically. The rates of CO2 production by the resting cell preparations were independent of the previous # values and had the same value in all three (10.4mmolesg -1 h-l). The specific rates of growth and CO2 production in the growing preparations were similar to those of the original cultures. These results seemed to indicate that intracellular factors control the specific rates of growth and of glucose transfer and that this control is lost in resting cells.
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Arch. Microbiol., Vol. 113 (1977)
A
"7
10
~c i c~
E E
"~o..
5
O u
9 . . . .
I 0.100
j 0.150
,% ~ o - i q~
- -o--
0.200
,u (h -I)
Fig. 5. Variation of the specific rate of non-fermentative CO2 production by Saccharomyces cerevisiae in 12 batch cultures at 25~ in mineral medium with glucose and vitamins
Relation between the Production of Non-Fermentative C02 and the Variation of the Specific Growth Rate Another difference between the resting and growing cells was the production of non-fermentative COz. In resting cells the molar amounts of ethanol and CO2 produced were identical, showing that all CO2 resulted from alcoholic fermentation. In growing cells the production of CO2 exceeded the production of ethanol. The production of non-fermentative CO2 depended on the specific growth rate (Fig. 5). At high values of/~ (0.23 h-1 _ 0.19 h-a), the specific rate of non-fermentative CO2 production was low and nearly constant (0.4 mmoles g-a h-a). At lower values of # the correlation with the specific rate of non-fermentative CO2 production was negative, the latter increasing to a value of 5.1 mmoles g-1 h-a at the lowest experimental g value (0.102 h-a), i.e. 34% of the total specific rate of CO2 production.
DISCUSSION The finding that the distribution of the specific growth rate in our strain of Saccharomyces cerevisiae was normal suggests that many factors, additive and independent in occurrence (Sokal and Rohlf, 1966) determine its variation. Neither the nature of these factors nor their primary targets have been identified. Bortels (1951) and his co-workers have reported on correlations between the rates of many microbial activities, including growth, and meteorological conditions. We became aware of his work after the conclusion of our experiments which had not been planned in a way that permitted to search a posteriori for such correlations. The influence of the history of the inoculum on the subsequent specific growth rate, as well as the lack of effect of washing the cells of exponentially
growing cultures on their specific growth rates, suggest that a number of possible physiological states may be established, which predetermine the specific growth rate during an extended period of time. In other words, each of these pysiological states appears to have some build-in inertia. This inertia is conceivably an expression of resistance to change by control mechanisms calibrated to maintain a given # value, imposed earlier on the cell population by unidentified factors. This concept implies that the observed variations of the yield (Ygluo) and of the specific rate of glucose (kgluc) were consequences rather than causes of the variation of the specific growth rate. Formal treatment of the variation of yg],c led to an estimate of kin, the maintenance coefficient, which was about six times higher than an estimate of k,, obtained earlier (van Uden, 1971) for the same strain in a glucose-limited chemostat culture in the same basal medium: 4.7 mmoles g-1 h-~ as compared with 0.8 mmoles g-a h-t. Thus, if the treatment is indeed applicable the conclusions would be, firstly, that maintenance requirements are substantially higher in batch cultures than in carbon-limited chemostat cultures and, secondly, that in batch cultures the maintenance requirements may be high enough to affect, measurably, the values of the yield. That this may well be the case was made more likely by the fact that y ~ x calculated for the set of batch cultures was very similar with the estimate obtained earlier (van Uden, 1971) for the same strain in the chemostat: 16.9 g mol -a as compared with 15.3 g mol-t. Similar relations were encountered (de Vries et al., 1970; Stouthamer and Bettenhausen, 1973) with a strain of Lactobacillus casei: the maintenance coefficient calculated from batch culture data was about three times higher than the estimate obtained in chemostat cultures. The notion that the variation of the specific growth rate determined the variation of the specific transfer rate of glucose, rather than the other way round, received support from the finding that control of kg]uc was lost when growing cells were forcibly changed into resting cells by eliminating nitrogen nutrient from the medium. Feed-back control of glucose transport in Saccharomyces cerevisiae is thought to be effected by an intracellular metabolite (Sols et al., 1971 ; Serrano and de la Fuente, 1974) which has tentatively been identified as glucose-6-phosphate (Azam and Kotyk, 1969; Becker and Betz, 1972). Our finding of the negative correlation between the specific growth rate and the specific rate of production of non-fermentative COz points to glucose-6-phosphate as a regulator. Increases in the pool size of this compound might simultaneously increase non-fermentative CO2 production through stimulation of the hexose monophosphate pathway
J. Martinez-Peinado and N. van Uden: Growth Rate Variation in Saccharornyces cerevisiae
and depress the transfer of glucose into the cell through feed-back inhibition of its transport. In yeasts, as in other microorganisms, the R N A content of the cells increase with increasing specific growth rate. Based on R N A measurements in S. cerevisiae by McMurrough and Rose (1967), we calculated an approximate ribose content of 220 Ixmoles per gram of dry weight of cells growing at a specific rate of 0.24 h -s, the highest/~ value we obtained. This corresponds to a specific rate of ribose production of about 53 gmoles g-~ h -~. The lowest specific rate of non-fermentative CO2 production we obtained (400 gmoles g-1 h-S) exceeds this maximum need by about one order of magnitude. Thus the observed negative correlation between the specific rates of growth and of non-fermentative CO2 production, does not conflict which the need for increased ribose production at higher specific growth rates. A possible explanation of the negative correlation may reside in the catabolism of carbohydrate reserves which occurs shortly before budding (yon Meyenburg, 1969; Kfienzi and Fiechter, 1969), the extent of which was negatively correlated with the specific growth rate. Though at least part of the reserves were catabolised by alcoholic fermentation, the data of those authors do not exclude a significant contribution of the hexose monophosphate pathway. Though the observed correlations between the variation of the specific growth rate and other parameters of growth may be of interest in themselves, the main finding consists in the observed isothermic variation of the specific growth rate under conditions of nutrient saturation. We conclude that #maxas defined by Monod (1942) is not a constant in the strain we studied, as it was not in the strain of Candida utilis by Stanley (1964). It remains to be seen whether this behavior is of general occurrence among microorganisms.
REFERENCES Azam, F., Kotyk, A. : Glucose-6-P as regulator of monosaccharide transport in baker's yeast. FEBS Letters 2, 333-335 (1969)
307
Becker, J. V., Betz, A.: Membrane transport as controlling pace. maker in Saccharomyces carlsbergensis. Biochim. biophys. Acta (Amst.) 274, 584-597 (1972) Bergmeyer, H. U. : Methoden der enzymatischen Analyse. Weinheim: Verlag Chemic 1962 Bortels, H. : Beziehungen zwischen Witterungsablauf, physikalischchemischen Reaktionen, biologischem Geschehen und Sonnenaktivit/it. Naturwissenschaften 38, 165 - 176 (1951) K~ienzi, M. T., Fiechter, A. : Changes in carbohydrate composition and trehatose-activity during the budding cycle of Saecharomyces cerevisiae. Arch. Mikrobiol. 64, 396-407 (1969) McMurrough, I., Rose, A. H. : Effect of growth rate and substrate limitation on the composition and structure of the cell wall of Saccharomyces cerevisiae. Biochem. J. 105, 189-203 (1967) von Meyenburg, H. K. : Energetics of the budding cycle of Saccharomyees eerevisiae during glucose limited aerobic growth. Arch. Mikrobiol. 66, 289-303 (1969) Monod, J_ : Recherches sur la croissance des cultures bact6riennes. Paris: Herman 1942 Pitt, S. J. : The maintenance energy of bacteria in growing cultures. Proc. roy. Soc. B 163, 224-231 (1965) Pirt, S. J. : Principles of microbe and cell cultivation. Oxford: Blackwell 1975 Serrano, R., de la Fuente, G. : Regulatory properties of the constitutive hexose transport in Saecharomyces cerevisiae. Molec. Cell. Biochem. 6, 161-171 (1974) Sokal, R. R., Rohlf, F. J. : Biometry. San Francisco: Freeman 1966 Sols, A., Gancedo, C., de la Fuente, G. : Energy yielding metabolism in yeast. In: The yeast, Vol. II (A. H. Rose, J. S. Harrison, eds.), pp. 271-307. London-New York: Academic Press 1971 Stanley, P. E. : The growth of Torulopsis utilis in aerated batch and continuous cultures. Ph.D. Thesis, University of Bristol (1964) Stouthamer, A. H., Bettenhaussen, C. : Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim. biophys. Acta (Amst.) 301, 53-70 (1973) van Uden, N. : Transport limited growth in the chemostat and its competitive inhibition; a theoretical treatment. Arch. Mikrobiol. 58, 145-154 (1967a) van Uden, N. : Transport limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch. MikrobioL 58, 155-166 (1967b) van Uden, N. : Kinetics and energetics of yeast growth. In: The yeasts, Vol. II (A. H. Rose, J. S. Harrison, eds.), pp. 75-118. London-New York: Academic Press 1971 de Vries, W., Kapteijn, W. M. C., van der Beck, E. G., Stouthamer, A . H . : Molar growth yields and fermentation balances of Lactobacillus easei L3 in batch cultures and in continuous cultures. J. gen. Microbiol. 63, 333-345 (1970) Umbreit, W. W., Burris, R. H., Stauffer, J. F. : Manometric techniques, 4th ed. Minneapolis, Minnesota: Burgess 1964
Received January 21, 1977
