Radiorespirometry of Candida utilis in phased culture under nitrogen-, carbon-, and phosphorus-limited growth

The release of 14CO2 from specifically labelled glucose ([G-1-14C],[G-2-14C],[G-3,4-14C], and [G-6-14C]) by phased cells of C. utilis was examined at intervals during 6-h cycles under conditions of N-,P-, and C-limited growth. On the basis that the release of 14CO2 from [G-1-C14] could serve as a measure of hexose monophosphate pathway (HMP) activity, of 14CO2 from [G-3,4-14C] as a measure of Embden-Meyerhof-Parnas (EMP) activity, and 14CO2 from [G-6-14C] as indicative of tricarboxylic acid (TCA) cycle activity, it was concluded that a relatively uniform operation of EMP occurred throughout the cycle in N-, P-, and C-limited cells, and of HMP and TCA in C-limited cells, but considerable variations took place in HMP and TCA cycle activities in N- and P-limited cells. The patterns of 14CO2 released from [G-2-14C] were more closely related to those observed from [G-1-14C] and [G-3,4-14C] than from [G-6-14C]. Changes in the rate of 14CO2 evolution during the cycle were sometimes abrupt and likely coincided with 'critical points' of metabolic activity in the cycle.     

Changes in phosphorus magnetic resonance spectra during the cell cycle of phosphorus limited phased culture of Candida utilis

Cell extracts, serially obtained from Candida utilis grown in continuous (synchrony) culture under phosphate limitation during an 8-h cycle and examined by NMR spectroscopy, revealed changes in polyphosphate content during the cycle period: other phosphorus containing components showed relatively little change. Initially zero, the polyphosphate content increased rapidly to a maximum after 30 min that coincided with exhaustion of phosphate from the culture, and then decreased slowly back to zero at the end of the cycle. The results suggest that polyphosphate, usually considered to function as a reserve material, actively participates during the cell cycle.     

A pilot plant apparatus for continuous phased culture--some observations on oxygen usage by Candida utilis during the cell cycle

An inexpensive scale-up of the cyclone column apparatus (Can. J. Microbiol., 11 , 893 (1965)) is described which is suitable for pilot plant studies on the 10 liter scale. Details of its construction and operation are given. Experimental with phased cultures of C. utilis growing in the equipment are reported during which studies of dissolved oxygen and oxygen uptake were made over the cell cycle. Changes in both oxygen parameters occurred during the cycle and were useful as monitors of the growth. Cell of C. utilis were found to be very sensitive to changes in oxygen supply during the early stages of the cell cycle.     

Relationship between iron-limited growth and energy limitation during phased cultivation of Candida utilis

The yeast Candida utilis was continuously synchronized by the phasing technique (6 h doubling time) with either iron or nitrogen as the limiting nutrient. Iron limitations resulted in decreased molar growth yields with respect to the carbon substrates and ammonia and in increased specific rates of oxygen uptake. Relatively low energy-charge values were maintained by the iron-limited culture. All these taken together seemed to indicate that the growth of the yeast under iron limitation was also limited by metabolically available energy. Consideralbe amounts of ethyl acetate were produced by the yeast under phased cultivation when the growth was limited by iron but not by nitrogen. In vitro studies using cell-free extracts showed that the substrates for ethyl acetate synthesis were acetyl coenzyme A (acetyl CoA) and ethanol. Under iron-limited growth acetyl CoA seemed to be diverted to ethyl acetate formation rather than being oxidized through the tricarboxylic acid (TCA) cycle. The possibility of energy limitation under iron-limited growth being brought about by the reduced capacity of the yeast to oxidize acetyl CoA through the TCA cycle is considered.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Cyclic accumulation of zinc by Candida utilis during growth in batch culture

Intracellular accumulation of zinc by Candida utilis NRRL-Y-7634 was mediated by an energy-and temperature-dependent, highly specific process exhibiting saturation kinetics. In zinc-supplemented medium, uptake occured only during the lad and late-exponential phases; this type of transport did not occur with zinc in bacteria nor with iron in either yeast or bacteria. Cells of C. utilis did not possess a zinc-efflux system; they could reduce their level of intracellular zinc only by dilution of the metal into daughter cells. Zinc-deficient organisms accumulated 12 times more zinc than did cells of the same culture age grown in zinc-supplemented medium. The varied, but experimentally reproducible levels of intracellular zinc that occured in response to the physiological and environmental parameters had no detectable effects on respiration, rate of growth, total cell yield, or cell viability. Neither the mechanism underlying the cyclic accumulation of sinc nor the function of such behaviour are understood.     

Changes in pattern of respiration and glucose utilisation in Candida utilis during the cell cycle: some variations with growth rate.

The release of 14CO2 from 14C-labelled glucose(G-1(-14)C, G-3,4(-14)C, G-6(-14)C) was followed in phased cultures of Candida utilis grown in a glucose- mineral salts medium under altered conditions of carbon:nitrogen limitation at doubling times of 2,4 and 6, h. Changes in oxygen uptake and CO2 evolution were observed and respirometric studies showed that the relative contributions of the Embden-Meyerhof-Parnas and hexose monophosphate pathways varied over the cell cycle and changed with growth rate. The results are discussed in relation to the growth metabolism of the cells.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

Extended culture: The growth of Candida utilis at controlled acetate concentrations

Extended culture, a special type of semicontinuous culture, permits prolonged maintenance of a constant or programmed environment in a growing culture by a controlled addition of one or more substrates. Differences between extended culture and continuous culture data are a measure of differences in the properties of cell populations with different cell age distributions but identical steady-state environments. Both extended culture and continuous culture were used to study the growth kinetics of Candida utilis (ATCC 9226) under conditions of substrate inhibition at controlled concentrations of sodium acetate in a carbon-limited mineral salts medium supplemented with 0.01 g/1 yeast extract. Acetate concentrations ranged from 1.2 g/l to 10.8 g/l (expressed as acetic acid), while yeast concentrations varied from 0.3 to 7.8 (g dry cells)/1. Rate parameters such as growth yields (Y), specific growth rates (μ), and linear growth rates (K), were calculated by computer from the data and theory presented herein. Specific growth rates as high as 0.54/hr were observed, although extended culture growth was more nearly linear than exponential in these experiments. Growth yields usually varied between 0.2 and 0.4 (g dry cells)/(g acetate), although values were as high as 0.8 for a brief period during one experiment. Growth yields at a given acetate concentration were correlated by an equation of the form 1/Y = 1/Y_G + m/μ. A maintenance coefficient (m) of 0.17 (g acetate)/(g dry cell-hr) was observed at acetate concentrations of 4.5 and 10. g/1. A typical maximum growth yield (Y_G) of 0.51 (g dry cell)/(g acetate) was obtained at 4.5 g/1 acetate, but an unusually high Y_G of 1.33 was found at 10. g/1 acetate. Oxygen uptake measurements are compared with these cell yield measurements. Linear growth rates in expended culture were correlated by the equation K = 0.89–0.70 (S/S₀) where K has units of (g dry cell)/(l-hr), S is the instantaneous acetate concentration, and S₀ is the initial acetate concentration. The extended culture kinetic data are shown to be substantially different from continuous culture kinetic data. Reason for these differences are discussed in light of diffrences in the cell age distributions, as well as possible differences in experimental conditions.     

Effect of pH on the rate of Candida utilis cell cycle initiation.

The rate of cell cycle initiation (as determined by the rate of bud emergence) in yeast Candida utilis under ammonium-limited phased cultivation was dependent on the pH at which the yeast was grown.     

Population study of cell cycle in a continuous culture ofCandida utilis

The mean lengths of G1, S, G2 and M phases of the cell cycle were determined on the basis of the population distribution ofCandida utilis grown in a continuous culture under steady-state conditions by using an original mathematical method. The length of the G2 phase was proportional to that of G1; the length of M was effectively independent of the growth rate. The length of S was proportional to the mean number of mitochondria in the cell.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Simultaneous action of environmental factors on Candida utilis growth in a chemostat culture

The effect of carbon dioxide and carbon-containing metabolites on the growth of Candida utilis was studied under the conditions of phosphate limitation. The limiting factor and the hydrocarbonate form of CO2 were shown to act simultaneously on the yeast growth, decreasing its rate. The threshold concentration of the limiting factor remained unchanged. Carbon-containing metabolites produced a similar action on the chemostat yeast culture. The factors limiting the rate of the yeast growth did not switch over.     

An antimycin A- and cyanide-resistant variant of Candida utilis arising during copper-limited growth

1. During copper-limited growth of Candida utilis in continuous culture on a non-fermentable carbon and energy source there is a selective pressure favouring the emergence of variants that are less dependent on copper. 2. We describe the properties of such a variant that by-passes cytochrome oxidase (EC 1.9.3.1) by utilizing an alternative oxidase communicating with the respiratory chain at about the level of cytochrome b. 3. Both direct studies of isolated mitochondria and calculations based on growth parameters showed that only one of the normal three phosphorylation sites was active. This site was localized between NADH and the cytochromes. 4. Growth of the variant with copper-supplemented media resulted in the return of cytochrome oxidase but not the loss of the alternative oxidase. 5. The alternative oxidase is inhibited by substituted benzhydroxamic acids. 6. Submitochondrial particles from the variant did not exhibit any novel electron-paramagnetic-resonance-spectroscopy features at about g=2.0 either at 80 degrees K or 12 degrees K.     

Changes in acid soluble nucleotide components of candida utilis during the cell cycle

Acid soluble extracts obtained at 30 min intervals from cells of C. utilis growing in synchrony in a phased culture (cycle time 51/2 hr) were fractionated on a Dowex-1-formate column. The series of fractionation profiles showed changes in number and amounts of components over the cell cycle. Transient accumulations of numerous components over the complex pool were observed. The significance of the changes are discussed in relation to practical applications and cell metabolism.     

He-Ne laser irradiation of folate-producing Candida utilis and optimization of culture conditions under submerged fermentation

In an attempt to obtain a microbial strain with higher yield of folate for industrial applications, we mutated the wild strain Candida utilis Y1.0 using a novel mutagenic process, i.e., irradiation by a helium–neon (He-Ne) laser with an output power of 20 mW and an exposure time of 20 min. The yield of folate in the mutated cells reached 1,102 ng/mL, which was 20.4-fold that of the wild strain. The mutant strain Y3.636 was relatively stable in terms of folate production through eight successive transfers of cultures and batch fermentation in a 3.7-L stirred-tank fermenter. Optimization further increased the yield of the mutant by 110 %, i.e., to 2,314?±?13 ng/mL. The optimal culture conditions for folate production were: cultivation in fermentation culture medium composed of 62.5 g/L glucose, 15 g/L corn liquor, 3 g/L (NH₄)₂SO₄, 3 g/L MgSO₄, and 1 g/L glutamic acid; inoculum size of 9 %; incubation at 28 °C and 196 rpm for 36 h. A time-course study of cell growth and folate production by mutant strain Y3.636 strongly suggested that folate production in C. utilis is growth-associated.     

Several parameters of the growth of chemostatic culture of Candida utilis in the presence of optimal and submaximal temperatures

Candida utilis BKM Y-1668 was cultivated in the chemostat (limitation with glycerol) with the rate of flow D from 0.05 to 0.3 hr-1; the economic coefficient Y and Ks were constant at the optimum temperature of growth (30 degrees C). The maximum growth rate was 0.35 hr-1. The content of ATP in the cells and the energy charge of the cell decreased, and the content of ADP and AMP and the activity of phoshohydrolases increased in the cell, with an increase in D from 0.05 to 0.3 hr-1. Small amounts of glycerol and phosphorus were expended for maintaining life without multiplication (m) at 30 degrees C. At the submaximum temperature (40 degrees C), growth of the cells was inhibited, the rate of assimilation of glycerol and phosphorus, and m, increased. The content of ATP in the cells and their energy charge also increased.