External factors limiting the multiplication potential of Tetrahymena

The rate at which a cell population expands under natural conditions is restricted by external factors, i.e. the multiplication rate of unicellular organisms, as well as of cell populations in multicellular organisms, can be enhanced by experimental means (Baserga, 1976). The limitation of growth rate and the termination of cell proliferation are essential factors in ontogenesis. Although the number of cell divisions may be limited in somatic tissues and in cell populations, this limit will be rarely attained but, rather, external factors will terminate cell division and/or induce sexual processes. The nature of the factors that limit the multiplication rate or terminate cell division under natural conditions is virtually unknown. Experimental studies testing the effects of external factors deal mostly with one or a few factors and evaluate either the growth rate of the culture or the final cell density. The present investigation tries to provide a comprehensive quantitative description of the limiting factors, starting from a set of standard external parameters for reference. This can only be done using a cell system for which the culture conditions are fully defined and are as reproducible as for the ciliate Tetrahymena. In Tetrahymena the effects of nutrition (Phelps, 1936; Prescott, 1957a; Curds & Cockburne, 1968; Cameron, 1973) or certain nutritional factors (Dewey, 1944; Dewey & Kidder, 1958; Chen, 1970; Evans, 1978, 1979 a, b, c;Evans & Witty, 1979; Evans et al. 1979) on the multiplication rate and final cell density have been measured. The results of these studies and their relation to the present results will be discussed later. The effect of temperature (Prescott, 19576; MacKenzie, Stone & Prescott, 1966; Cameron & Nachtwey, 1967; Cleffmann, 1967) and hydrostatic pressure (Zimmerman, 1969) on the culture growth have also been described and will not be included in this investigation.

The factors tested here are : cell density, oxygen supply, pH, undefined conditioning factors, metabolic products, proportional changes in all constituents of the medium, and of certain groups of nutrients. The criteria by which the capacity for multiplication is evaluated are: maximal density, duration of exponential multiplication phase, and multiplication rate during this phase.

Tetrahymena thermophila, strain B, was maintained in the vegetative growth phase. Stock cultures of 15 ml were kept at room temperature in large test tubes.

The defined medium was first described by Kidder & Dewey (1951). It was later supplemented with cholesterol and proteose peptone (cf. Everhardt, 1972) and contains the following components in mg/1.

The chemicals were from Merck, Darmstadt, W. Germany. Components at less than 1 mg/1 were dissolved at 100-fold concentration and added accordingly. Glucose was dissolved at 100-fold concentration, sterilized separately, and added after sterilization. Water for media was prepared by a Milli-Q-System (Millipore, Bedford, Mass. U.S.A.) in which the water passes successively through a charcoal column, 2 ion-exchange columns, and a filter. Media prepared in this Water yield the same growth parameters as those prepared with double quartz-distilled water. The medium was autoclaved 40 min at 200 kPa (= 116 °C). The pH of the medium has to be adjusted with M-NaOH to 7·2. The medium was stored at 6 °C for not longer than 3 weeks.

Experimental cultures contained either 250 ml in 1-l Erlenmeyer flasks or 100 ml in 500-ml Erlenmeyer flasks. They were shaken at 29 °C at a speed that was just too slow to generate foam.

Cultures were started in prewarmed medium mostly at a density of 1000 cells/ml from a culture in exponential growth phase. Inoculation and sampling were performed under sterile conditions using an inoculation hood.

Different additions to the medium such as nutritional substances and NH₄⁺ were tested for their effects on culture growth. These substances were dissolved at high concentrations in sterile water and small volumes were added to the cell suspension. The resulting change in cell density was corrected for.

Samples were taken immediately after inoculation and at intervals of 1–3 h at the start and at the end of exponential growth phase. Cultures were shaken thoroughly to assure an even distribution of cells at sampling. Cell density was determined in a Coulter Counter BZ. Repeated sampling from the same culture yield a standard deviation of less than 2% of the mean. Parallel cultures yield growth curves that are almost identical (cf. Fig. 3). In many cases differences are smaller than the width of the symbol in the figures.

The end of the exponential growth phase was determined by the calculation of the doubling time between each 2 points. These times were plotted against cell density. The end of exponential growth was set at the point at which this curve exceeded the doubling time of 210 min.

Content of NH, in the medium was determined according to Berthelot (Bemt & Bergmeyer, 1970), referring to an ammonia standard. O₂ concentration was determined in the culture using an O₂ electrode (WTW Weilheim, Germany).

Experiments were designed to find out whether the medium of an old culture has any properties that reduce or enhance the growth potential of a culture. Such properties could be withdrawal of essential nutrients or secretion of growth-inhibiting or growth-promoting factors. The existence of growth-promoting factors is unlikely because the growth curve in that case should exhibit an increasing growth rate whereas it is, indeed, constant.

Cells from late log phase and stationary cultures were eliminated by centrifugation (3000 rev./min, 5 °C, 10 min). The conditioned medium was used to start a new culture with log-phase cells at low density. The growth curve of these cultures (Fig. 1) shows clearly that the growth-sustaining capacity of the conditioned medium is greatly reduced. Cultures started with medium from late log phase grew with a doubling time of about 10 h as compared to less than 3 h in control cultures. In addition the culture growth was terminated after 2–3 doublings. These figures vary widely because the properties of the medium change rapidly at the end of the exponential growth phase.

Medium from stationary cultures did not allow any increase in cell number if a new culture was inoculated at low density.

These data suggest that the medium of a stationary culture is either depleted of some nutritional components or contains metabolic products in concentrations that inhibit culture growth. To support this result further cultures with a density of 10⁵/ml were diluted by 50%. This was done in parallel with fresh medium and with isosmotic salt solution (Fig. 2).

Both sets of experimental cultures continued to grow at the same rate after dilution. The final cell density in the cultures diluted without nutrients was precisely half of that in the control culture. In cultures to which fresh medium had been added the cell density reached that of the control. It can be concluded from these data that neither the cell density per se nor the concentration of inhibiting metabolic products cause the termination of culture growth or affect the growth rate.

If the depletion of all or some nutrients is responsible for reduction in growth rate and termination of culture growth, it should be possible to extend culture growth by fortifying the medium with additional nutrients. Therefore, 25 ml of a 10-fold concentrated complete medium were added to a 250 ml culture in late log phase. The maximal cell density in this culture containing twice the normal concentration of nutrients was twice that of the control culture. If this was repeated twice, the cell density increased repeatedly (Fig. 3). The maximal cell number was proportional to the amount of nutrients (Table 1).

Nitrogen derived from amino acids is excreted as ammonia in Tetrahymena (Hill & van Eys, 1965). We were not able to detect any urea in the culture medium. Products of nucleic acid metabolism are found (e.g. hypoxanthin) but are not considered in this study. The concentration of ammonia in the pure medium varied between 0·1 and 0·4 μmol/1. The variation apparently depends on different batches of proteose peptone. During culture growth the concentration rose to about 1·0μmol/1 (Fig. 4).

The concentration is closely related to cell density. After addition of further nutrients, and thereby increasing cell density, the ammonia concentration increased to 1·3 μmol /1 (data not shown).

To test the effects of ammonia we increased the concentration of ammonia from the beginning of the culture by replacing sodium acetate with ammonium acetate to obtain NH₃ concentrations of 1·0μmol/and 1·8μmol NH₃/, respectively. Cultures with the lower concentration exhibited a lag at the beginning and a reduction of exponential phase, but the growth rate and final density were the same as in the controls. With the higher concentration one observes, in addition, a slight reduction in growth rate (Fig. 5). Nitrogenous metabolic products such as ammonia or urea, therefore, are not responsible for termination of culture growth and limitation of growth rate. They may, at higher concentrations, reduce the multiplication rate at the end of exponential phase.

The pH can be excluded as a growth-limiting factor. The normal pH range of a growing culture is in the range from 7·2 to 7·4. It rises slightly during culture growth but does not exceed 7·4 as long as the cell number increases. During extended stationary phase the pH rises to about 7·8. As Prescott (1958) has shown (for a different species of Tetrahymena but with the same medium), the growth rate is reduced by not more than 5% by alteration of pH within the range of 6·6 to 7·6.

We have tested oxygen concentration as a possible limiting factor by increasing and reducing the oxygen supply. These experiments have been performed with a Tetrahymena sp. (formerly designated as T. pyriformis HSM). Under standard conditions this strain yields a reduced cell density of 230000/ml and the doubling time of the cell number is 210 min. All other growth characteristics were the same. Three parallel cultures were either agitated as described above, or additionally aerated, or neither agitated nor aerated. In these cultures cell density and O₂ concentration were determined simultaneously (Fig. 6) (M. Rübsam, unpublished results). The O₂ concentration is different, as expected. Increase of oxygen supply had little effect on cell yield. In stagnant (neither agitated nor aerated) cultures the growth rate was reduced already when the density came to 20000/ml. Nevertheless, the same density as in control cultures was finally reached. These results partially confirm the findings of Levy & Scherbaum (1965). It is evident that under the present standard conditions the termination of culture growth is not due to an effect of lack of oxygen, because the cultures cease at an oxygen concentration at which the stagnant cultures are still growing exponentially. The data also show that O₂ concentrations lower than 3 mg/1 effectively reduce the growth rate of the culture.

The results described so far demonstrate that under the conditions of this study the amount of nutrients is the factor that determines the limitation of culture growth. Since the medium is almost completely chemically defined we did experiments to determine which of the components are growth-limiting. We added groups of the nutrients, to a final doubling in concentration, to the standard culture, and determined the growth characteristics of the culture.

The following groups were tested: amino acids, nucleosides, glucose, vitamins and salts, proteose peptone, medium without proteose peptone, and medium without proteose peptone and amino acids.

Table 2 presents the results. Doubling of the concentration of proteose peptone caused a 27% increase in cell density. Doubling of the concentration of the total medium except proteose peptone, on the other hand, increased the cell density by 70%.

Doubling of amino acids alone increased cell yield by about 20% whereas doubling of all nutrients except nitrogen sources had no effect. This indicates that peptone and amino acids are the limiting factors but the effects of the 3 groups: proteose peptone, amino acid, and the rest of the medium, are not additive. Proteose peptone and amino acid effects together cannot account for the growth potential of the complete medium. Doubling amino acids together with all non-nitrogenous compounds (all nutrients except proteose peptone) on the other hand has a higher growth-promoting potential than amino acids alone. This indicates that after increasing nitrogen sources other components become growth-limiting.

Table 2 also presents the length of the exponential growth phase and the doubling time of the cultures during exponential phase. It is evident that addition of vitamins, salts, and nucleosides had no effect. Addition of glucose gave a slightly higher cell density. The lengths of the exponential phase are in accord with the results for cell density.

It should be noted that none of the alterations of culture conditions described in this study had any effect on the rate of increase in cell number during log phase.

Since it is known that Tetrahymena grows and multiplies on axenic media (Lwoff, 1923), several modifications of defined media have been described. The composition was basically worked out by Kidder & Dewey (1951). Elliot, Brownell & Gross (1954) described the recipe used in this study (cf. Everhardt, 1972). Tetrahymena cultures grow well on complete synthetic medium although the growth rate is low and varies. Addition of cholesterol and proteose peptone increases the growth rate considerably and makes it highly reproducible. This enriched synthetic medium, together with optimal temperature (29 °C), optimal pH (7·25), and oxygen supplied by agitation, is widely used for studies of cell metabolism when defined nutrition conditions are required. Therefore, these conditions have been chosen as a basis for investigating the significance of external factors for culture growth.

In this situation Tetrahymena cells multiply exponentially as long as all the nutritional components are available. Exhaustion of one or more of them reduces the multiplication rate and subsequently terminates culture growth. Addition of nutrients extends the exponential growth and increases the final cell density. Similar results (Phelps, 1936; Curds & Cockbume, 1968; Cameron, 1973) demonstrate that the final cell density also depends on the amount of food under different conditions.

The present data prove that the final cell density is proportional to the amount of nutrients. This is evident from the effect of dilution of the medium (Fig. 2) and of additional supply (Fig. 3). From this proportionality it can be concluded that no other factor limits culture growth. It is not known what cell density eventually can be reached by increasing the amount of synthetic medium, but information from the literature and from this laboratory suggests that cell density cannot be increased much beyond 1·5 × 10⁶/ml, not even with rich organic media.

The growth rate of the culture is not increased by higher concentrations of the nutrients constituting the defined medium. The doubling time of 180 min, however, is not the lower limit, since it can be reduced to about 120 min, e.g. in organic media.

From the results of the present experiments it has been ruled out that other external factors such as O₂ concentration, pH, concentration of metabolic products, or cell density per se limit multiplication rate and terminate culture growth. All of these factors of course may eventually inhibit cell multiplication (as indicated by Figs. 5 and 6) but the limiting concentrations are well beyond the range of a normal culture. In particular, there are no known conditioning factors produced by the cells, whether inhibiting or promoting, The conditioning factors, described by Kidder (1941) and Saitoh & Asai (1980), are found using rich organic media and by different experimental procedures. The results of these authors may also be explained by their experimental approaches.

From the 30 components constituting the enriched synthetic medium, the compounds serving as nitrogen sources are the ones that limit culture growth. Cell density can be increased by amino acids as well as by proteose peptone. All other components, like nucleic acid precursors, vitamins and salts, are present in excess and therefore have no effect (cf. Evans, 1978, 1979 a-c). The cell yield in the presence of amino acids together with the other non-protein nutrients, however, is higher than with amino acids alone. This suggests that with the addition of amino acids some other factors become limiting. This explains why Evans & Witty (1979) obtained no increase in cell density by addition of amino acids in T. pyriformis strain W, since the con-centration of amino acids is up to 3 times higher in their medium than in that used in this study. The enriched synthetic medium, therefore, can be considered well balanced and a significant extension of culture growth can be achieved only by increasing the concentration of all ingredients.

This work has been supported by the Deutsche Forschungsgemeinschaft, Sonderfor-schungsbereich 103 ‘Zellenergetik und Zelldifferenzierung’.