Yeast Candida Utilis Research Articles

Hyperaccumulation of zinc by zinc-depleted Candida utilis grown in chemostat culture

The steady-state levels of zinc in Candida utilis yeast grown in continuous culture under conditions of zinc limitations are <1nmol Zn2+/mg dry weight of cells. Unlike carbon-limited cells, zinc-depleted cells from a zinc-limited chemostat possess the capacity to accumulate and store zinc at levels far in excess of the steady-state level of 4 nmol/mg dry biomass observed in carbon-limited chemostat cultures. Zinc uptake is energy-dependent and apparently undirectional since accumulated 65Zn neither exists from preloaded cells nor exchanges with cold Zn2+. The transport system exhibits a high affinity for Zn2+ (Km =.36micrM) with a Vmaxof 2.2 nmol per minute per milligram dry weight of cells. Growth during the period of the uptake assay is responsible for the apparent plateau level of 35 nmol Zn2+/mg dry weight of cells achieved after 20-30 min in the presence of 65Zn at pH 4.5 and 30 degrees C. Inhibition of growth during the uptake assay by cycloheximide results in a biphasic linear pattern of zinc accumulation where the cellular zinc is about 60 nmol/mg dry weight after 1 h. The enhanced level of accumulated zinc is not inhibtory to growth. Zinc-depleted C. utilis contains elevated amounts of polyphosphate and this anionic evidence does not allow discrimination between possible regulation of zinc homestasis either by inhibitions of zinc efflux through control of the membrane carrier or by control of the synthesis of a cytoplasmic zinc-sequestering macromolecule.

Physiology of Candida utilis yeast in zinc-limited chemostat culture.

Candida utilis NRRLY -900 was grown in aerobic continuous culture in a minimal salts medium with sucrose (1% w/v) as the carbon source. increasing the concentration of zinc in the medium from 2.3 microM results in an increase in the apparent critical dilution rate from 0.3 to 0.47h-1, and in the maximum biomass productivity form 1.5g dry weight per litre per hour (at D=0.33-1) to 2.56g per litre per hour (at D = 0.45-1). The maximum steady-state level of cell-associated zinc (at D = 0.4-1) is 4nmol Zn2+/mg dry weight, during carbon-limited growth, and about 9mumol Zn2+/mg dry weight when FeCI3 is omitted form the medium. At input zinc concentrations <20microM, the cellular zinc concentration decreases linearly in proportion to the input zinc concentration and at <4.5microM Zn2+ the culture becomes zinc-limited. Zinc-limitation results in a decrease iun the growth yield Ysurcosefrom 0.54 to 0.38 cells/g sucrose consumed, and Y0 from 22 to 13g cells/g-atom 02, suggesting an altered efficiency of energy metabolism. The composition of the culture biomass with respect to protein and RNA content is not affected by zinc limitation.

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.

Relationship between cell size and efficiency of synchronization during nitrogen-limited phased cultivation of Candida utilis.

Under the phased method of cultivation the yeast Candida utilis grew and divided synchronously. The newly formed cells were relatively small, and a new cell cycle was not initiated until the cells could attain a certain minimum size (critical size). Although the cells expanded to some extent after division, the critical size was not reached until a fresh supply of medium was provided. With the arrival of the fresh supply of growth medium at the beginning of the phasing period, the cells expanded rapidly, and new cell cycles were initiated. The cells continued to expand until the growth-limiting nutrient (nitrogen source) was exhausted or until 90 min, which ever occurred first. Usually, buds emerged at a constant time after the start of the phasing period. The time of bud emergence was independent of the size attained by the cells during the expansion phase of growth. The results indicated that it was initiation of the cell cycle that was under size control, and not bud emergence. Bud emergence seemed to be under the control of a timer. The start of this timer seemed to be at or immediately after the beginning of the phasing period. Protein synthesis was essential for the initiation and expansion of buds. However, inhibition of protein synthesis by cycloheximide did not prevent unbudded cells or the parent portion of budded cells from expanding. Cycloheximide seemed to abolish the control mechanism(s) which prevented the cells from expanding after they had reached the maximum size.

Single Cell Protein Recovery from Alfalfa Process Wastes

ABSTRACT CULTIVATION of the yeast Candida utilis on waste brown juice obtained from a pilot-scale process for mechanical extraction of alfalfa protein gave high biomass conversion yields with significant reductions in total dissolved solids and organic carbon levels. Protein content of the yeast product was improved considerably by supplementing the brown juice with nitrogen and phosphorus.

Elimination of ethanol inhibition of yeast growth by a multistream ethanol feed in a multistage tower fermenter

AbstractThe effect of several types of ethanol feed into a multistage tower fermenter, under conditions of oxygen supply rate limitation, on growth and physiological activity of the yeast Candida utilis was studied. The measurements were made at steady states of continuous culture for single values of dilution rate, temperature and pH in all stages of the fermenter and with a constant total sum of ethanol supplied. It was found that the type of ethanol feed significantly affects both the economic parameters associated with biomass production and the cell physiology in the individual stages of the fermenter.

Induction effect of PO 2 during continuous yeast production from ethanol in a multistage tower fermentor

The effect of the periodic variation of the partial pressure of oxygen in the aeration gas on biomass concentrations, ethanol conversion, yield and productivity during continuous cultivations of the yeast Candida utilis in a multistage tower fermentor was studied. The results were compared with those obtained under aeration conditions with a constant PO 2 in the aeration gas. The results demonstrated that, with the optimum PO 2 in the aeration gas, the aeration procedure with the periodic variation of PO 2 in the gas phase permitted achievement of the same process parameters as those under constant PO 2. Using this new aeration procedure, the consumption of pure oxygen can be lowered by 55% to 60%. In addition, the significance of the induction effect of PO 2 on growth characteristics in the individual stages of the fermentor was proved.

Microbial Production of Methyl Ketones. Purification and Properties of a Secondary Alcohol Dehydrogenase from Yeast

Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding agents.

Respiratory oscillations and heat evolution in synchronous cultures of Candida utilis.

Synchronous cultures of the budding yeast Candida utilis prepared by continuous-flow size selection showed respiratory oscillations when the energy source was either glucose, acetate or glycerol. The period of the oscillations was about one-third of the cell cycle time (i.e. about 0.5 h). No fluctuations in heat evolution could be detected. In organisms growing with acetate or glycerol, the effects of cyanide, N,N'-dicyclohexylcarbodi-imide and carbonyl cyanide m-chlorophenylhydrazone (maximum inhibition of respiration at respiratory maxima, maximum uncoupling of energy conservation at respiratory minima) suggest that the control mechanism responsible for the oscillations is mitochondrial respiratory control in vivo. The effects of cyanide and N,N'-dicyclohexylcarbodi-imide on the respiration of cultures growing synchronously with glucose were different from those for cultures growing with the non-fermentable substrates; this suggests that the mitochondrial respiratory system interacts with the early reactions of glucose utilization.

Effect of PO2 on growth and physiological characteristics of Candida utilis in a multistage tower fermentor

AbstractThe effect of increasing the partial pressure of oxygen in the aeration gas on growth and physiological activity of the yeast Candida utilis in a multistage tower fermentor was studied. The measurements were made at steady states of continuous culture for single values of dilution rate, temperature, and pH in all stages of the fermentor and with one given ethanol concentration in the growth medium feed. The partial pressure of oxygen in the gas phase was changed in the range from 165 to 310 torr. The results revealed the existence of the upper critical value of the partial oxygen pressure in the gas phase. It was demonstrated that the upper critical value of PO2 influences not only the growth rate, biomass yield, and productivity but also the cell physiology resulting in changes of respiration activity and activity of alcohol and aldehyde dehydrogenases.

A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms.

The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor. The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.

Growth characteristics of Candida utilis in a multistage culture system

The effect of increasing ethanol concentration in the feed on the growth and physiological activity of the yeast Candida utilis was studied. At steady-state of continuous culture with constant values of dilution rate, temperature, and pH in all fermenters in series biomass, ethanol and volatile acid concentrations, biomass yield and productivity and respiration activity were measured. In the three-stage system the maximum biomass concentration in the effluent and maximum productivity was achieved between 20 and 25 g ethanol/l in the feed. At higher concentrations, ethanol negatively affects the coupling of oxidative phosphorylation and respiratory control of cells resulting in a decrease in biomass yield and intracellular protein content. It was shown that the presence of acetate in the medium inhibits the respiration activity of yeasts growing on ethanol.

Comparison of yeast and algal diets for bivalve molluscs

Four species of bivalve molluscs were fed diets consisting of varying proportions of the yeast Candida utilis and the diatom Thalassiosira pseudonana. Juvenile Argopecten irradians, Mercenaria mercenaria, and Mytilus edulis grew as fast or faster than controls when fed diets containing as much as 50% yeast. Growth of soft tissue in Crassostrea virginica, however, decreased with the amount of yeast in the diet. The relative food values of the different diets were not closely correlated with gross chemical composition or amino acid composition.

Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuliger on potato processing wastewater.

The yeasts Candida utilis and Saccharomycopsis fibuliger were propagated as a source of single-cell protein in a continuous, mixed, aerobic, single-stage cultivation on blancher water generated during potato processing. A series of steady-state experiments based on a two-level factorial design, half-replicate modified with an intermediate experiment, was performed to determine the effect of pH, 3.8 to 4.8; dissolved oxygen, 42 to 80% saturation; dilution rate, 0.17 to 0.31 h(-1); and temperature, 27 to 32 degrees C on the amount of carbon consumed, the rate of carbon consumption (R(c)), the amount of reducing sugar consumed, the rate of sugar consumption (R(g)), the amount of protein produced, the rate of protein production (R(p)), the yield from carbon, and the yield from reducing sugar. The results were analyzed by stepwise multiple regression and Fisher's least significant difference test. Analyses showed that high dilution rates resulted in increased R(c), R(g), and R(p) and indicated that a rate of 0.31 h(-1) was below the critical dilution rate. A temperature of 32 degrees C increased the amount of carbon consumed by 34%. A pH of 4.3 to 4.8 increased the amount of protein produced. The yield from carbon was constant, and the relatively high yield from reducing sugar indicated that other substrates were consumed. Dissolved oxygen was in excess at 42% saturation and above. Since C. utilis predominated the mixed cultures and amylase production appeared to be limited, a single-stage fermentation lacked efficiency. The experimental design allowed preliminary optimization of major environmental variables with relatively few experiments and provided a basis for future kinetic studies.

Functional properties of yeast grown on ethyl alcohol

AbstractA new series of food ingredients, based on torula yeast (Candida utilis) grown on ethyl alcohol, offers unique functional properties and flavor effects in processed and fabricated foods. The new ingredients bind fat and water, act as emulsifiers, heighten meat, poultry, and seasoning flavors, improve texture of bakery products, and add crispness and tensile strength to wafers. At the same time, they contribute desirable nutrition to foods by increasing the quantity and improving the quality of protein in cereals.

Differential extraction of soluble pools from the cytosol and the vacuoles of yeast (Candida utilis) using DEAE-dextran

The plasma membrane of Candida utilis cells was rapidly disrupted by a small dose of DEAE-dextran. The vacuolar membranes, in contrast, remained intact under isotonic conditions. Therefore, the cytosolic pool could be extracted in a first step, and in a second step, after disruption of the vacuoles, the vacuolar pool. The two extracts were studied in cells grown on different nitrogen sources, namely ammonium, arginine, ornithine, citrulline, glycine, and proline.

Toxic effects of fatty acids on yeast cells: possible mechanisms of action.

As shown in a previous paper, threshold concentrations of lower and intermediate fatty acids inhibit the uptake of inorganic phosphate, growth, and cell division in yeast cells. This demonstrates that, apart from these effects, the acids cause an increase in the respiration quotient (RQ), inhibition of CO2 fixation, production of ethanol at the expense of anabolic processes, and inhibition of active amino acid transport in the yeast Candida utilis. On the other hand, the threshold concentrations have no effect on intracellular pH. The inhibition of the inorganic phosphate uptake cannot be the sole primary mode of action of fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought about by fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought by fatty acids at concentrations still premitting some phosphate uptake. Although 2,4-dinitrophenol and caproic acid at low concentrations cause an analogous decrease in biomass yield, their combination does not bring about any marked increase in the effect. Considering the physicochemical properties of fatty acids and their preferential action on energy-requiring processes, one of the key sites of action can be assumed to be the mitochondrial membrane. Fatty acids might inhibit the transport of anions, especially phosphate, across the membrane, and disturb the membrane potential by affecting the transport protons. The physiocochemical properties of fatty acids may also give rise to their binding to other intracellular membranes and to a subsequent interference with the function of the corresponding organelles.

A general method for characterizing naturally occurring folate compounds, illustrated by characterizing Torula yeast ( Candida utilis) folates

A method previously found suitable for the chromatographic separation and identification of simpler folates has been extended and found suitable for separating and characterizing all the complex polyglutamyl folyl derivatives occurring naturally. Folates were characterized employing the combined use of analytical DEAE-cellulose chromatography, folylpoly-γ-glutamyl carboxypeptidase digestion, and differential microbiological growth response studies. An observed relation between the log phosphate concentration of the eluting buffer and the number of γ-glutamyl residues in successive pteroylpoly-γ-glutamyl derivatives provides a simple tool for a rapid and accurate identification of folate compounds from their elution profile. Twelve folate compunds present in Torula yeast ( Candida utilis) were characterized employing this method; 80% of the total folates appeared to be pteroylpolyglutamates. The method characterizes not only the number of γ-glutamyl residues but also the state of reduction of the pteridine ring and the nature of the 1-C substituents attached.

Rapid purification of threonyl-tRNA synthetase from Saccharomyces carlsbergensis by affinity elution from phosphocellulose.

Threonyl-tRNA synthetase [EC 6.1.1.3] of Saccharomyces carlsbergensis was highly purified by a simple enzyme-substrate affinity method. After calcium-gel treatment of the crude enzyme extract, ammonium sulfate precipitation, and removal of polynucleotides with DEAE-cellulose, the enzyme was nonspecifically adsorbed onto phosphocellulose (P-cellulose), and then eluted specifically with a linear concentration gradient of threonine tRNA from torula yeast (Candida (Torulopsis) utilis). The enzyme was purified 303-fold from the calcium-gel supernatant. This purification method is very simple and time-saving. The purified enzyme gave a single band on SDS-gel electrophoresis, indicating a molecular weight of 82,000, and exhibited a molecular weight of about 150,000 by gel filtration. This suggests that the enzyme consists of two identical subunits. Some kinetic properties of the pure enzyme are described.

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.