[Use of rifampicin for directed modification of the nucleotide composition of the cells of Bacillus megaterium and Candida utilis]. / Primenenie rifampitsina dlia napravlennogo izmeneniia nukleotidnogo sostava kletok Bacillus megaterium i Candida utilis.

The object of this work was to study the effect of rifampicin on the physiological state of Bacillus megaterium and Candida utilis in chemostat and batch cultures. When B. megaterium is cultivated in a medium containing 1 microgram of rifampicin per litre, its metabolism changes, the pool of free nucleotides rises, and the content of RNA drops in response to the specific action of the inhibitor. The modified state is observed throughout many generations. The action of the inhibitor is also non-specific: protein content falls and polyhydroxybutyric acid is accumulated. The morphology of B. megaterium cells changes: they become larger, their cell wall thickens, and the separation of cells from one another is complicated. At a high concentration (5 mg/l), rifampicin decelerates the growth of C. utilis and causes merely a minor non-specific inhibition.

[Cytology of Candida utilis yeasts under different temperature regimes of continuous cultivation]. / Tsitologiia drozhzhei Candida utilis pri razlichnykh temperaturnykh rezhimakh nepreryvnogo kul'tivirovaniia.

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.

[Chemostat cultivation of Candida utilis yeasts under nonstationary temperature conditions]. / Khemostatnoe kul'tivirovanie drozhzhei Candida utilis pri nestatsionarnykh temperaturnykh usloviiakh.

The yeast Candida utilis VKMY-1668 was cultivated in the chemostat (D = 0,34 h-1) while the temperature was changed many times from 31 to 37 degrees C at a frequency of 2 h (one generation). Control experiments were conducted at the constant optimal (31 degrees C) or supraoptimal (37 degrees C) temperatures. Changes in the biochemical composition and morphology of the yeast were assayed as well as electro-kinetic characteristics of the cell surface. All of the above properties changed periodically with a change in the growth temperature. This occurred however only at the first stage of the nonstationary temperature regime. Later, changes in the temperature from 31 to 37 degrees C had no effect on the content of RNA, protein and amino acids in the biomass or on the electro-kinetic characteristics of the cells. Apparently, the nonstationary temperature regime was responsible for stationary chemostat cultivation, similar to the growth at the constant optimal temperature of 31 degrees C (despite changes in the temperature from 31 to 37 degrees C). Protein content in the yeast was the most stable parameter while RNA content changed in the biomass in the course of growth.

[Periodic changes in the morphophysiological properties of chemostat culture of Candida utilis yeasts under alternating temperatures]. / Periodicheskie izmeneniia morfofiziologicheskikh svoistv khemostatnoi kul'tury drozhzhei Candida utilis v usloviiakh peremennykh temperatur.

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.

[Yeast growth under alternating temperature conditions and the possibility of originating thermotolerant forms]. / Rost drozhzhei v usloviiakh peremennykh temperatur i vozmozhnost' proiskhozhdeniia termotolerantnykh form.

The chemostat culture of Candida utilis was grown under the conditions of varying temperatures: one generation grew at an optimal temperature of 31 degrees C, the second at supraoptimal temperatures: either 35, 36 or 37 degrees C; these were alternated during 6-8 generations. Changes in the growth yield were studied as well as changes in the content of protein, RNA and DNA in cells and changes in the zeta-potential of cells at a growth rate of 0.34 hr-1. At this rate, the population became "synchronous-like" and contained up to 60% of simultaneously budding cells. When the temperature of cultivation was periodically changed, the content of RNA and DNA in the biomass fluctuated at a decreasing amplitude around values found during the growth at the optimal temperature; the electro-kinetic characteristics of cells were also in the attenuated oscillating state approaching the norm. The authors discuss how the population retains the value of growth yield constant when it is grown under the conditions of varying temperatures and what is the origin of thermotolerant forms.

[Method of calculating the specific growth rate of microorganisms by measurements of the substrate or product concentration]. / Metod vychisleniia udel'noi skorosti rosta mikroorganizmov po izmereniiam kontsentratsii substrata ili produkta.

A procedure and formulae are proposed for calculating the specific rate of microbial growth; these are based on measuring the concentration of either a limiting substrate or a product involved in the growth of a microorganism. Only the concentrations of a substrate (S) or a product (P) are determined periodically in the course of the cultural growth whereas the biomass yield (chi) in the medium is established only after long time intervals. The proposed equations may be used for controlling the specific growth rate in large-scale processes by the concentration of a product (or a substrate) using a computer, as well as in laboratory studies of mu as a function of the conditions for growth of slowly growing microorganisms.

[Electrokinetic properties of microorganisms as dependent on culture conditions]. / Elektrokineticheskie svoistva mikroorganizmov v zavisimosti ot uslovii kul'tivirovaniia.

The electrokinetic properties of cells were studied with the batch and continuous cultures of Bacillus megaterium and the chemostat culture of Candida utilis. The above cells growing at a higher rate had a higher electrophoretic mobility and a greater negative zeta potential; this seems to stem from the accumulation of negatively charged groups on their surface. The bacterial cells had higher values of electrophoretic mobility and electrokinetic potential comparing to the yeast cells; the electrokinetic properties of the bacterial cells changed more with a variation of the growth rate than in the yeast cells.

[Effect of the protein synthesis inhibitor chloramphenicol on the basic growth indices of continuous and periodic Bacillus megaterium cultures]. / Vliianie ingibitora sinteza belka khloramfenikola na osnov nye pokazateli rosta nepreryvnoi periodicheskoi kul'tury Bacillus megaterium.

The action of chloramphenicol, a specific inhibitor of bacterial growth and an inhibitor of protein synthesis, was studied in the conditions of continuous and batch cultivation. Steady states of the population within the range of D from 0.2 to 0.7 hr-1 were obtained at a concentration of the antibiotic equal to 20 microgram per liter. The shape of the chemostat curve in the presence of chloramphenicol in the medium indicates that the antibiotic slightly decreases mu max and considerably diminished Y. A change of limiting factors occurs along the chemostat curve at different dilution rates (0.2, 0.4 and 0.7 hr-1): one-, two- and three-factor effects on the population were observed. Steady states of the population in the chemostat are possible by chloramphenicol concentrations of 10 to 100 microgram per litre; in this case, cellular metabolism changes in the following nonspecific way: less effective energy processes are activated, the Y decreases as well as protein content in the cells, but the content of poly-beta-hydroxybutyric acid in the biomass increases. If chloramphenicol is taken at a concentration of 1000 microgram per litre (a dose strongly inhibiting the growth rate), other changes specific for this inhibitor are found: the content of protein decreases while that of RNA increases, and substances of nucleotide nature are released into the medium. The changes remain for many generations. The data show that it is possible to change the ratio between cell polymers in growing population.

[Nonspecific stress in Candida utilis under growth inhibition by a specific inhibitor]. / Nespetsificheskii stress u Candida utilis pri ingibirovanii rosta spetsificheskim ingibitorom.

The physiological state of Candida utilis chemostat cultures was studied when the growth was limited with glycerol at D from 0.047 to 0.35 hr-1, and compared with the same cultures inhibited with cycloheximide. This compound at a concentration of 1 mg/litre almost did not inhibit the growth, but the value of the growth yield decreased from 0.45 to 0.38; the content of protein fell, on the average, from 44 to 35% of the dry biomass weight; the content of polysaccharides increased from 22 to 30%; the content of nucleic acids and lipids did not change; the liberation of keto and amino acids into the medium slightly increased. The population became heterogeneous, the cells increased in size, the separation of daughter cells from the parent ones became complicated, and the number of vacuoles in the cells increased. All of these changes, apart from a significant decrease in the content of protein, are also typical of the action of various nonspecific growth inhibitors.

[Cell adhesion in a chemostat culture of Candida utilis under the influence of supraoptimal temperature and elevated acidity]. / Adgeziia kletok v khemostatnoi kul'ture Candida utilis pod vliianiem supraoptimal'noi temperatury i povyshennoi kislotnosti.

The effect of the supraoptimal temperature (38, 40 degrees C) on the chemostat culture of Candida utilis was studied. The above factor caused a part of the biomass to float as a thin layer of foam to the surface of the medium. After an hour, the concentration of the cells on the surface could be four times as high as that within the medium. The content of protein was the same in the cells taken from the surface and from the depth. Singular cells or their groups (2 or 4--8 cells) were found deep in the medium whereas cells on the surface were aggregated forming conglomerates of 20--100 and more cells. The simultaneous action of the elevated tmperature and the acid pH value made flotation of cells onto the surface more stable and protracted (it could be maintained in a chemostat for weeks).

[Principle growth indices of a chemostat Candida utilis culture resistant to acid pH values]. / Osnovnye pokazateli rosta khemostatnoi kul'tury Candida utilis, ustoichivoi k kislym znacheniiam pH.

The kinetics of growth of the Candida utilis chemostat culture 1668-3-37 was studied in a synthetic medium with ethanol at different values of pH and temperature. Chemostat curves were obtained for the pH of the medium of 4.5 and 3.0 and the temperature of 32 degrees C. The following growth characteristics were determined: the maximal growth rate (mumax), the economical coefficient (Y), the substrate (saturation) constant (Ks), the rate of ethanol uptake (q), maintenance energy (m). The minimal amount of ethanol inhibiting the yeast growth was assayed in short-term experiments under periodic conditions with shaking. The value of mumax was 0.35 hr-1 when the yeast was cultivated at 32 degrees C and the pH 4.5, and 0.32 hr-1 at the pH 3.0. The value of Ks varied by an order of magnitude at different pH values when the chemostat culture was grown at D=0.2 hr-1: 36.0 mg/litre at the pH of 3.0 and 3.75 mg/litre at the pH of 4.5. The value of m was close to 0 at the pH of 4.5 and equaled 5--7 mg of ethanol per gram of dry biomass per hour at the pH of 3.0; it was still higher when the temperature of cultivation was increased to 38 degrees C. The minimal substrate (ethanol) concentration inhibiting the yeast growth was constant at different cultivation conditions (pH 3.0 or 4.5 and temperature 32 or 38 degrees C), being equal to 0.45% (v/v) of ethanol.

[Effect of heat shock on the amino acid makeup of the whole cells and walls of Candida utilis yeasts in chemostat cultivation]. / Vliianie teplovogo shoka na aminokislotnyi sostav tselykh kletok i stenok drozhzhei Candida utilis pri khemostatnom kul'tivirovanii.

The amino acid composition of intact cells and cell walls was determined in Candida utilis AUCMY-1,668 growing in the regime of chemostat with limitation by glycerol or ethanol deficiency at a temperature of 30 degrees C (control) or with inhibition by an elevated temperature of 40 degrees C (experiment). In the control, intact cells contained 43-44% of amino acids, and cell walls, about 10% (per the weight of dry cell walls); the following amino acids prevailed in the cell walls: threonine, glutamic acid, serine and leucine. The content of amino acids decreased in both the intact cells and cell walls at the elevated temperature (40 degrees C). The content of leucine, methionine, tyrosine and cystine decreased in the cell walls more than in the intact cells (with regard to the total amino acid content of the cell walls and intact cells, respectively). Under the action of the elevated temperature, the cells became larger and did not separate: the scar formed at the end of budding stretched between the mother and daughter cells holding them together.

[Effect of raised temperatures on the protein and RNA synthesis rate in yeasts]. / Vliianie povyshennykh temperatur na skorost' sinteza belka i RNK u drozhzhei.

The rates of incorporation of labelled precursors of protein (14C-DL-leucine) and RNA (14C-uracil) into the cells of synchronous yeast cultures of Pichia membranaefaciens, Hansenula anomala, Saccharomyces cerevisiae and Saccharomyces (fabospora) fragilis were studied at different temperatures. Synthesis of RNA and then of protein was inhibited in H. anomala if the temperature was increased above the optimal one. This is manifested even more distinctly in Sacch. cerevisiae and P. membranaefaciens. In the thermotolerant yeast Sacch. fragilis, the incorporation of 14C-uracil was inhibited at temperatures above 40 degrees C while the rate of protein synthesis did not decrease.

[Effect of increased temperatures on RNA and protein synthesis in the cells of a synchronous Candida utilis culture]. / Vliianie povyshennykh temperatur na sintez RNK i belka v kletkakh sinkhronnoi kul'tury Candida utilis.

The rate of incorporation of labeled precursors for RNA ([14C]uracil) and protein ([14C]DL-leucine) into the cells of the synchronous culture of Candida utilis VKMY-1668 (the optimum temperature of growth, 31--32 degrees C) was studied as a function of different temperatures (28, 31, 32, 34, 36, 38, and 41 decrees C). The yeast was grown on a simple mineral medium containing glycerol. RNA synthesis was found to be more susceptible to elevated temperature than protein synthesis: the maximum rate of incorporation was registered at 32--34 degrees C for [14C]DL-leucine and only at 32 degrees C for [14C]uracil (the rate of its incorporation at 34 degrees C decreased by 50% as compared to that at 32 degrees C). The rate of incorporation of [14C]uracil at 34 degrees C reached 100% (the rate at 32 degrees C) when yeast autolysate was added to the medium, and 75 and 70%, respectively, upon the addition of DL-methionine or Mg2+ (as compared to 50% without them).

[Effect of raised temperature on the developmental cycle of Candida utilis yeasts]. / Vliianie povyshennoi temperatury na tsikl razvitiia drozhzhei Candida utilis.

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.

[Effect of elevated temperature on the morphology of a chemostat culture of Candida utilis yeasts and on their content of nucleic acids]. / Vliianie povyshennoi temperatury na morfologiiu khemostatnoi kul'tury drozhzhei Candida utilis i na soderzhanie v nikh nukleinovykh kislot.

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).