Replacement of Potassium Ions by Ammonium Ions in Potassium-Limited Chemostat Cultures of Candida Utilis NCYC

FEMSLE 05264

Replacement of potassium ions by ammonium ions in potassium-limited chemostat cultures Downloaded from https://academic.oup.com/femsle/article/107/1/1/627950 by guest on 27 September 2021 of Candida utilis NCYC 321

Ed T. Buurman 1, Paul T.D. Herman, M. Joost Teixeira de Mattos and Oense M. Neijssel

Department of Microbiology, Biotechnology Centre, Unicersity of Amsterdam, Amsterdam, Netherlands

(Received 20 October 1992; revision received and accepted 12 November 1992)

Abstract: Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.

Key words: Candida utilis; Potassium; Ammonium; pH; Chemostat culture

Introduction gests that ammonium ions can contribute significantly to the osmotic pressure of the cytoplasm. It Prokaryotes require a high intracellular con- has been proposed that a high intracellular am- centration of potassium ions in order to activate monium ion concentration could be built up, be- the ribosomes [1] and to maintain the osmotic cause the ammonia molecule is extremely perme- pressure of the cytoplasm [2]. Recently it has able through the bacterial membrane. This causes become clear that under some growth conditions a concentration gradient of ammonium ions across ammonium ions can to a large extent take over the membrane (as described by the Henderson- the physiological role of potassium ions in Hasselbalch equation) which is dependent solely prokaryotes [3]. This confirms the early observa- upon the pH gradient across this membrane [3]. tions that ammonium ions can activate enzymes Research on the potassium requirement of requiring potassium ions [4,5] and further sug- Candida utilis growing in chemostat cuture [6] has shown that the potassium content of cells growing in a potassium-limited culture was much lower Correspondence to: O.M. Neiissel, Department of Microbiol- (i.e. 0.15% w/w) than that of similarly-grown ogy, Biotechnology Centre, University of Amsterdam, P.O. Box 20245, 1000 AE Amsterdam, Netherlands. prokaryotic cells, such as Klebsiella aerogenes 1 Present address: Department of Molecular Genetics and (0.8% w/w) or Bacillus subtilus var. niger (2.7% Cell Biology, University of Chicago, Chicago, Illinois, USA. w/w). Furthermore, it was shown that the potassium ion concentration effected the efficiency of Analyses oxidative phosphorylation in mitochondria iso- Cellular protein content was determined using lated from C. utilis [6]. Finally, the same authors the biuret method and using bovine serum albu- concluded that potassium ions could be replaced min (Sigma) as a standard [9]. Calculations and by rubidium ions, but not by ammonium ions [7]. all other analyses were as described previously Since there is no a priori reason to presume [3]. that yeast membranes are less permeable towards the ammonia molecule, we reinvestigated whether

Results Downloaded from https://academic.oup.com/femsle/article/107/1/1/627950 by guest on 27 September 2021 ammonium ions could replace potassium ions in C. utilis and, if so, under what circumstances this Candida utilis was grown in potassium-limited occurred, and the results are reported below. chemostat culture with either ammonium chloride or sodium nitrate as the nitrogen source. The steady-state dry weight decreased with increased Materials and Methods dilution rates, indicating that cells required more potasssium when growing at a high rate. This Organism and growth conditions confirms the results of Aiking and Tempest [6]. Candida utilis NCYC 321 was maintained by As the dry weight increased proportionally with monthly subculture on tryptic meat-digest agar the potassium chloride concentration in the slants and grown in a Modular Fermenter 500 medium, the yield on potassium chloride (YK, the Series (LH Engineering Co. Ltd., Stoke Poges, amount of dry weight per input amount of potas- Bucks UK) or in a Porton-type chemostat [8]. sium chloride) remained constant (Fig. 1A). From Media and growth conditions were as described this it can be concluded that these cultures were previously [6] except that 100 mM NH4C1 was truly potassium-limited and that the potassium used, and that at low culture pH values 2 N HC1 concentration in the extracellular fluids at the was used as titrant. imposed growth rates was relatively low.

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Dilution rate (h") Culture pH value Fig. 1. Yield on potassium chloride (YK, g dry weight per mmol potassium chloride) of potassium-limited chemostat cultures of Candida utilis (t = 30°C); grown at a constant culture pH value of 5.5 at various dilution rates (A) or grown at a constant dilution rate of 0.1 h -1 at various culture pH values (B). The nitrogen source was either 100 mM NH4CI (closed symbols) or 100 mM NaNO 3 (open symbols). The input KC1 concentration was 100/xM (circles), 200/zM (triangles) or 400/~M (squares). At a growth rate of 0.1 h-1 the dry weight of potassium ion, which puts previous observations potassium limited cultures was threefold higher on the potassium requirement of this organism in when ammonium chloride (100 raM) was used as a different perspective. Moreover, the results de- nitrogen source as compared to cultures grown scribed above are not a trivial extension of previ- with sodium nitrate (Fig. 1A). A lowering of the ous observations made with prokaryotes, because ammonium chloride input concentration caused a eukaryote contains different intracellular com- the dry weight to decrease and at an input con- partments, each possibly possessing its own pH centration of 30 mM the dry weight was riot value and/or potassium ion concentration, which significantly different front that of a similar cul- leads to a number of complications. Downloaded from https://academic.oup.com/femsle/article/107/1/1/627950 by guest on 27 September 2021 ture grown with nitrate as the nitrogen source Firstly, the permeability towards ammonia of (data not shown). the cell membrane and the membranes of the In order to verify that the increase in dry different cell organelles may be different. As a weight in the presence of ammonium ions was result, it may be impossible to increase the am- due to a lowered potassium requirement and not monium ion concentration in some or all cell to an accumulation of macromolecules, the over- organelles (including the cytoplasm) sufficiently all cell composition was determined of potassium- to allow for a replacement of potassium ions by limited cells grown with either ammonium chlo- ammonium ions. Furthermore, the physiological ride or sodium nitrate as the sole nitrogen source. role of potassium ions in C. utilis could be strictly The cell composition varied with the growth rate, dependent on the presence of an ion gradient but no large differences between ammonium- and across the outer or one of the intracellular mem- nitrate-grown cells were observed (data not branes. The build-up of a similar gradient of shown). ammonium ions could require a much steeper pH In prokaryotes the extent of potassium re- gradient than is present across such a membrane placement was related to the extracellular con- in a viable cell. The similarity in behaviour be- centration of ammonia [3] which, of course, de- tween prokaryotes and this yeast is therefore pends both on the input ammonium chloride remarkable. concentration and the culture pH value. This In C. utilis the increase in dry weight occurred latter culture parameter was found to influence at ammonia concentrations much lower than those the requirement for potassium ions of C. utilis to causing an effect in K. pneumoniae (Fig. 2). This such an extent that at a culture pH value of 3.5 is due to the much lower potassium content of C. no difference in biomass concentration was ob- utilis, but not necessarily to a lower potassium served between potassium-limited cultures grown concentration in the potassium requiring com- with ammonium chloride (extracellular ammo- partment(s). When most of the potassium ions nium ion concentration in steady-state about 80 are required in a large compartment (e.g. the mM) or sodium nitrate as the nitrogen source cytoplasm), the potassium concentration will be (Fig. 1B). Again no difference in cell composition low. Therefore, only a low ammonium ion con- between ammonium- and nitrate grown ceils could centration in this compartment is required to be observed (data not shown). replace the potassium ions. However, when the bulk of the potassium ions would be concentrated in a small compartment, the ammonium ion con- Discussion centration has to be high at a relatively low ammonia concentration and thus require a rela- The results presented here are qualitatively tively high pH value in that compartment. highly comparable to those previously found with The apparent decrease of the effect of ammo- the prokaryotes Bacillus stearothermophilus and nium ions on the biomass concentration at higher Klebsiella pneumoniae [3]. It is therefore justified dilution rates (Fig. 1A) can be understood when to conclude that also in C. utilis the ammonium it is realized that on the one hand the require- ion can take over (some of) the role(s) of the ment of C. utilis for potassium equivalents in- 30 pest [7] found that with C. utilis grown under various nutrient limitations (but with excess ru- 25 bidium and potassium ions) the potassium content remained constant, whereas the relative rubidium content was strongly increased only under 2O nitrogen-limiting conditions (i.e. in the absence of ammonium ions). This suggests that under these >- 15 conditions a lack of ammonium ions was compen-

sated for by uptake of rubidium ions. Downloaded from https://academic.oup.com/femsle/article/107/1/1/627950 by guest on 27 September 2021 10 If one assumes that all potassium ions were located as free ions in a single compartment (with 5 a constant pH value), one can derive that the absolute contribution of ammonium ions to the

i i i i i i I i i total requirement of monovalent cations is pro- 0 1 2 3 4 5 6 7 8 9 10 portional to the prevailing ammonia concentration, irrespective of the contribution of potassium [NH 3] ions. Mathematically this implies a hyperbolic relationship between dry weight and ammonia Fig. 2. Yield on potassium (YK, g dry weight per mol potassium chloride) of potassium-limited chemostat cultures of concentration (at a constant potassium chloride Candida utilis (squares) (D= 0.1 h-l; t =30°C) and potas- input concentration). The fact that the yield on sium-limited chemostat cultures of Klebsiella pneumoniae potassium did not increase hyperbolically (Fig. 2) (circles) (D = 0.27 h-l; t = 35°C) at various ammonia concen- can be explained by assuming that the intracellu- trations. The ammonia concentration was varied by keeping lar ammonium ion concentration did not increase the culture pH value constant at 5.5 (C. utilis) or 8.0 (K. pneumoniae) at different ammonium chloride input concen- proportionally with the extracellular ammonia trations (open symbols) and by changing the culture pH value concentration. This could be due to an increase at a constant ammonium chloride input concentration (100 of intracellular pH value(s); alternatively, a signif- mM) (closed symbols). Ammonia concentration is given in icant amount of potassium ions could be present /zM for results of C. utilis and in mM for results of K. in a non-bound form and could only be replaced pneumoniae. competitively by higher intracellular ammonium ion concentrations. creases with increasing growth rate, but that on the other hand the ammonium ion concentration References remains constant. Thus, while at a growth rate of 0.1 h -1 ammonium ions constituted 67% of the 1 Tempest, D.W. (1969) Syrup. Soc. Gen. Microbiol 19, 87- total requirement (which can be derived from the 111. 2 Epstein, W. (1986) FEMS Microbiol. Rev. 39, 73-78. threefold increase in dry weight of ammonium- 3 Buurman, E.T., Pennock, J. Tempest, D.W., Teixeira de grown cells as compared to nitrate-grown cells Mattos, M.J. and Neijssel, O.M. (1989) Arch. Microbiol. (Fig. 1A)), at a growth rate of 0.3 h-1 they only 152, 58-63. contributed 25% of that percentage (i.e. 17%) 4 Suelter, C.H. (1970) Science 168, 789-795. due to the fourfold increase in the requirement 5 Pestka, S. (1972) Proc. Natl. Acad. Sci. USA 69, 624-628. 6 Aiking, H. and Tempest, D.W. (1976) Arch. Microbiol. 108, for monovalent cations. Similar calculations can 117-124. be made to explain why Aiking and Tempest [7] 7 Aiking, H. and Tempest, D.W. (1977) Arch. Microbiol. 115, concluded that ammonium ions could not replace 215-221. potassium ions in C. utilis: the ammonium chlo- 8 Herbert, D., Phipps, P.J. and Tempest, D.W. (1965) Lab. ride concentration in the medium that was used Pract. 14, 1150-1161. 9 Herbert, D. Phipps, P.J. and Strange, R.E. (1971) In: Meth- in these studies was too low to give a reasonable ods in Microbiology, Vol. 5B (Norris, J.R. and Ribbons, dry weight in the absence of added potassium. D.W., Eds.), pp. 209-334. Academic Press, London-New It is interesting to note that Aiking and Tem- York.