Search

Five anamorphous yeast strains of ascomycetous affinity with a specific mode of budding were isolated from raised bog soils of Bakcharskoe Bog (Tomsk oblast). According to their morphological and physiological properties, these strains belong to the genus Candida but differ from all species described previously. The level of DNA-DNA homology with species similar in the assimilation spectrum was as low as 7%. Based on these data, the new species Candida aurita sp. nov. is described.

The effect of the RNase from Bacillus intermedius on the growth and trophic cycle of Candida utilis was studied. The RNase at concentrations of 0.001-0.01 microgram/ml stimulated yeast growth by 30-40% as compared to the control, reduced the mitotic cycle of the yeast by shortening its G1 phase, and decreased the number of exotrophic cells in the G1 phase to a minimum. It was suggested that RNase is involved in the regulation of the transition of cells from the exo- to endotrophic state.

The effect of thiamine and biotin on the processes of cell division, assimilation of glucose, and accumulation of the biomass and nitrogen in the cells was studied with the Candida yeast. The action of the vitamins depended on the source of nitrogen. In some strains, asparagine can substitute for biotin. Biotin has different effect on the production of gamma-aminobutyric acid in Candida pulcherrima, C. guilliermondii. C. tropicalis K3-10. High concentrations of arginine were found in C. guilliermondii var. membranaefaciens in the presence of biotin. The vitamins did not favour the assimilation of nitrate nitrogen in species which were not adapted to this source of nitrogen.

Optimal conditions were found for the production and isolation of the protoplasts of Candida lipolytica. The maximum amount of the protoplasts was produced after 90 minutes of the incubation with a crude preparation of the enzyme from Helix pomatia (100 mg/g wet biomass). Longer incubation results in lysis of the protoplasts and structural damages of the intracellular components. The yield of the protoplasts does not depend on the nature of stabilizing agent. A decrease in the stabilizer concentration increases the yield of the protoplasts four times. Preliminary treatment of the yeast cells with a 0.1 M solution of SH compounds (cysteine, beta-mercaptoethanol) does not increase the yield of the protoplasts; and 0.2 M solution of these compounds decreased the yield of the protoplasts.

The effect of zinc ions on the physiological state of Candida utilis VKM Y-1668 was studied during its chemostat cultivation. The concentration of zinc ions was maintained at a constant level. Zinc was found to inhibit the growth at a concentration of 300 mg/l. The cells were rapidly washed from the fermenter when zinc concentration was increased to 500 mg/l. Zinc uptake by the culture became greater when zinc concentration in the medium was gradually increased. If the yeast was cultivated in the presence of the inhibitor, the content of protein, RNA, DNA and polysaccharides fell while lipid content increased. Growth inhibition by zinc ions interfered with cell division; the cells could not separate from one another and formed catenae and conglomerates.

Candida utilis daughter cells were obtained from a chemostat and their cell cycle was studied in batch cultures at a glucose concentration of 0.1, 1 and 2% in the medium. The size of the daughter cells growing at a 0.1% concentration of glucose reached only a half size of the cells growing at a 2% glucose concentration. Cytokinesis took place under the conditions of substrate limitation as soon as the cycle of DNA division was over and the resultant daughter cells were extremely small.

The object of this work was to find out whether it was possible to cultivate yeasts in chemostat in varying regime: at multiple changes to temperature from the optimal one (31 degrees C) to the supraoptimal one (37 degrees C) and back with a frequency comparable to the generation time; this regime was alternated during 32 generations. The cultures were compared with chemostat cultures grown in steady-state regimes at 31 or 37 degrees C. The value of Y, the composition of cells and morphometric characteristics were determined by the optical-structural computer analysis. The size and shape of cells and the optical properties of the protoplasm were found to be in the oscillatory regime correlating with changes in the growth temperature and periodically tending to the normal characteristics. The value of Y, the content of RNA and protein in the biomass gradually stabilized. A possibility, in principal, to continuously cultivate yeasts is discussed when the conditions of the environment are rhythmically changed and the process is controlled in the morpho-physiological characteristics of the cells.

The techniques of light and electron microscopy were used to study structural changes in Candida lipolytica cells related to limitation of their growth and a nitrogen source in medium with glucose and hexadecane and transition of the culture to the production of citric acids. Changes in the content of protein in the cell and in the activities of enzymes of the citrate and glyoxylate cycles located in mitochondria and peroxysomes of the yeast were investigated at the same time. The results characterizing cytological changes in the cell of C. lipolytica in the conditions of citric acid biosynthesis fit in well with the data on protein content and enzyme activities in the cells obtained by biochemical analysis. The transition of the yeast cells to the overproduction of citric acids has been shown to be related to profound rearrangements of the cell: the inhibition of protein synthesis; changes in the structure of the cytoplasm; the appearance of vacuoles of an irregular shape, often in contact with the mitochondria; changes in the mitochondrial and peroxysomal apparatus; the accumulation of lipids. It has been demonstrated once again that the peroxysomal apparatus of the yeast cell has a relationship with the metabolism of n-alkanes.

A population of Candida boidinii was grown under the chemostat conditions at a dilution rate of 0.14-0.20 h-1 in a mineral medium with methanol as a sole source of carbon and energy. Its age structure was analysed taking account of the reproductive age (the number of cycles) and the cyclic age of the cells (the phase of a cell cycle). Four morphological phases of the cycle, namely, one phase of preparation for budding and three successive budding phases, were compared with the phases G1, S, G2 and M. In terms of their reproductive age, the cells can be arranged as follows: (1) cells that formed no buds at all, 43 +/- 5%; (2) cells after one cycle of budding, 28 +/- 4%; (3) cells from which two and more daughter cells have separated, 29 +/- 4%. The age structure of an yeast population must be analysed when it is necessary to estimate its physiological heterogeneity or to elaborate the cultivation of microorganisms involving the control over the age structure of the population.

Cultivation of Candida tropicalis IBEM 303 without thermostatic regulation on n-alkanes results in an increase of the temperature in the fermenter to supraoptimal values and to the linear growth of the culture without distinct differentiation according to the growth phases. The cells grown at supraoptimal temperature differ from the cells cultivated at optimal temperature by the amount of some amino acids and oligopeptides liberated by the yeast into the cultural broth, by the chemical composition of the cells and their morphology.

The activity of alcohol oxidase (AO) in the cells of Candida boidinii, in actively growing cultures, depends on the stage of the cell cycle. The minimal activity of AO was found in young cells produced by budding and in cells in the process of budding. The maximal activity of AO was detected in the fraction of cells of a medium size. The activity of AO decreased abruptly when the cells proceeded to budding. The activity of catalase depended on the stage of the cell cycle only to a minor extent.

Candida utilis cells growing in one-step chemostat at D = 0.05, 0.1, 0.25 and 0.35 h--1 were separated into fractions of daughter cells (without scars) and parent cells (with scars). The daughter cells were transferred under the conditions of batch cultivation where the following parameters were determined: the duration of the maturation phase, the specific growth rate, and changes in the length, width and volume of the cells. The differences in the behaviour of various daughter cell types are discussed in terms of the significance of age distribution for characterizing the average physiological state of the yeast population.

A population of the yeast Candida boidinii growing in the chemostat in a medium with methanol was separated in dextran solutions of different density. The density of cells was found to vary from 1.04 to 1.11 g/cm3, being 1.08 g/cm3 on the average. Changes in the cell density correlated with variations in the content of water and dry matter in the cells and its density. Cell fractions of different density contained cells which mainly belonged to a certain phase in the cell cycle. However, the fractions were not homogeneous. The authors propose a scheme to account for changes in the cell density during the cell cycle. According to this scheme, within the cycle, the density of the cell rises twice (when the cell prepares for the budding and in the budding phase II) and drops down twice (in the budding phases I and III).

The growth of Candida guilliermondii on media with glucose and acetate is described by statistical functions for cell volume (size) distribution. Special instrument has been used to measure the volume of cells and volume distributions. Changes in the morphological structure of populations (both in the average size of cells and in the extent of the size heterogeneity) were detected throughout the whole period of the cultural growth including the exponential phase. These changes hardly differed during the growth on media with glucose or with acetate. The content of flavins in cells also varied in the course of periodic growth. Their total level in cells, as well as the level of FAD, was higher in the exponential phase of growth than during the subsequent phases. Changes in the intracellular level of flavins were found also in yeast populations synchronized by nitrogen limitation.

The effect of the submaximal temperature (41.5 degrees C) on growth was studied with a synchronous periodic yeast culture. If the cells were subjected to the action of elevated temperature at the beginning of the growth cycle, the formation of buds was not inhibited in contrast to the separation of nuclei between the daughter and parent cells. If the cells started their growth cycle at the optimal temperature of 32 degrees C and, after spending 0.6 of the cycle at this temperature, were subjected to a temperature of 41.5 degrees C, the separation of nuclei between the daughter and parent cells took place, but the cells were not entirely separated one from another.

The protoplasts of Candida utilis 295 t were produced with the aid of the lytic enzyme from Helix pomatia. If the cell wall of C. utilis 295 t is not treated with SH-compounds (the best effect was found with L-cysteine), it is resistant to the action of the enzyme. The yield of the protoplasts was 100 per cent after 15 minutes of the incubation with the lytic enzyme if the cells were preliminarily treated with L-cysteine. Optimal conditions for the production of the protoplasts are described.

The effect of elevated temperature (40 degrees C) on a chemostat culture of Candida utilis was studied at different rates of dilution, D = 0.1 and 0.3 hr-1. The cells in the fermenter being in the stationary state at the optimum temperature of 30 degrees C were gradually washed-out 5 hours after the action of this temperature, and the population consisted of non-divided cells. In the majority of such "double" cells, the nucleus was contained in both the parent and the daughter parts. The content of RNA decreased by 31%, that of DNA, by 20%, and that of protein, by 13% (per 1 mg of biomass).

The paper describes the conditions in which the spheroplasts of the yeast Torulopsis candida IBFM-Y-127 with a high respiration rate can be isolated. The preliminary incubation of the cells with SH-reagents has to be carried out in a buffer without an osmotic stabilizer, and the incubation in a medium containing 0.6 M KCL, 0.1 M MgSO4, 0.1 MKH2PO4, pH 5.2. In these conditions the cells are incubated with the enzyme from Helix pomatia during 15 to 20 minutes, and the yield of the spheroplasts is 95 to 100 per cent. The spheroplasts oxidize various substrates (glucose, acetate, ethanol) at the same, or even higher, rate as the intact cells.

Changes in the structure of cells and in the morphology of cytoplasmic membranes (CM) were studied when Candida utilis VKM-1668 was grown under the conditions of continuous cultivation at constant (31 or 37 degrees C) and varying temperatures. The structure of cells was studied using the technique of ultrathin sections, and the morphology of CM was investigated by the technique of freezing-etching. At the optimal temperature of growth, the structure of cells was found to be typical of the yeast while the CM was shown to have a folded surface with narrow slit-like invaginations. When the temperature was elevated, nuclear division and cell separation were inhibited, the dimensions of cells increased, and the slit-like invaginations of CM straightened out. At a varying temperature regime of cultivation, the structure of cells and the morphology of their CM were similar to those observed at the constant optimal temperature of growth.

The effect of temperature on the growth rate, morphology and ultrastructure of the yeast Candida utilis was studied under the conditions of turbidostat and chemostat. The mean size of the yeast cell was found to increase at suboptimal and supraoptimal temperatures while the quantity of membranous structures was shown to decrease under the same conditions. The mean size of cells grown under the conditions of turbidostat was much greater than that of cells grown under the conditions limited with glucose. The mechanisms involved in morphocytological changes at suboptimal and supraoptimal temperatures appear to be different.

The dimensions of daughter cells without scars, and of mother cells with 1--4 scars, of Saccharomyces cerevisiae and Candida utilis were studied in the one-stage chemostat at D equal to 0.05, 0.1, 0.25, and 0.35 hr-1. The average dimensions of the cells increased with the specific growth rate. At low values of the specific growth rate, "immature" daughter cells separate and attain reproductive ability as independent individual organisms. A twofold increase in the biomass of yeast cells occurs at the account of growth of the buds and small daughter cells.

Using the method of radioactive indicators, the specific content of proteins, lipids, total phosphorus, and lipid phosphorus was estimated in the morphological cell fractions of Candida tropicalis after growth in a liquid mineral medium containing n-octadecane as a source of carbon. The morphological cell fractions were produced by means of differential centrifuging. The following comparatively pure fractions were obtained simultaneously: soluble fraction, microsomes, mitochondria, large membranes, and cell walls.

The effect of growth inhibiting values of pH of the medium (pH 2.3 and 7.8) on physiological, morphological and cytological characteristics was studied with the chemostat culture of Candida utilis. Changes in the pH of the medium were accompanied with a decrease in the economic coefficient of the yeast cells, an increase in the respiratory quotient and the activity of phosphohydrolase. Phase-contrast micriscopy has shown that, at acid pH values, the cells were smaller than in the control (in the cells grown under optimal conditions) and rather uniform, oval or rounded, and contained large lipid granules. The presence of large lipid granules was confirmed by electron microscopy; vacuoles were smaller than in the control cells, but their number was greater than one; mitochondria stuck together and had one general outer membrane; the cell wall was 3--4 times thicker. At alkaline pH values of the medium, the whole cellular organization of C. utilis was disordered: the cells were larger, the size of vacuoles increased too much, and the structure became heterogeneous.

The effect of cyclic pH and pO2 changes on the morphology of Candida utilis was studied with time intervals measured in minutes. The pH was varied from 4.5 to 2.6, the pO2 from 70% of the saturation to 0%. The morphology was studied both visually and using the technique of optical-structural computer analysis. The regime with pH changes increased the biomass yield. However, the averaged morphological characteristics showed that the growth was slightly inhibited. Therefore, the yeast population was very heterogeneous, some cells were inhibited while other cells were stimulated, which made the economic coefficient rise. The cells were also inhibited when they were exposed in the conditions of oxygen deficiency for a short period of time. However, the cells could not be inhibited if, at the same time, the pH was extremely low. Changes in the morphology were detected earlier than either the inhibition or stimulation of growth was recorded by measuring the weight of biomass.