The production of extracellular polysaccharides by a hydrocarbon assimilating yeast.

Investigations were made on the extracellular polysaccharides production by a hydrocarbon assimilating yeast. The yeast Candida lipolytica was grown on two different media containing n-hexadecane as the sole carbon source. Polymeric materials precipitated from the culture medium with ethanol were determined gravimetrically at various growth periods. The hydrolysis of the precipitated material and their chromatographic analysis revealed the presence of mannose, glucose and galactose in the yeast extract-containing medium. The proportions of these sugars differed at various growth periods. The hydrolysates of the polymers of the yeast extract-free medium contained more xylose than those of the yeast extract containing medium.     

Utilization of kerosene by a hydrocarbon-assimilating strain of Fusarium oxisporum

Fusarium oxysporum IFO 6384 was grown in three different media, one containing glucose and two containing kerosene as carbon source. One of the kerosene media contained also Tween 60. The harvested dry mats were weighed at various stages of growth. Substitution of glucose with kerosene led to a sharp drop in the production of fungal mycelia. Analysis of the mat samples representing maximum yields indicated that the mycelia grown in the kerosene media were richer in free amino acid and poorer in protein as well as in polymeric and low-molecular weight carbohydrates than those grown in the glucose medium. Treatment of the culture filtrates with three volumes of acetone led to the separation of two fractions. In the first, acetone soluble fraction the free sugars and amino acids were determined, whereas the second, acetone precipitable one, was examined for its proteolytic activity. The gelatinase activity of the acetone precipitable material (APM) obtained from the kerosene media was lower than that from the glucose medium. The former activity, in contrast to the latter reached its maximum and minimum values at pHs 4 and 9, respectively.     

Effect of phospholipids from Saccharomyces cerevisiae at different stages of development on restoration of succinooxidase activity in lipid-depleted mitochondria

Phospholipids extracted fromSaccharomyces cerevisiae at different stages of development after glucose repression contain three major fatty acids: palmitic, palmitoleic and oleic. The ratio palmitic: palmitoleic strongly decreases beginning at the 6th hour of growth.To test the effect of fatty acid composition and in particular of unsaturation on succinoxidase activity, all these phospholipids, phospholipids from commercial yeast, and Asolectin were incubated with lipid-depleted yeast mitochondria. The amount of P bound was not much different for the various phospholipids; succinoxidase activity was restored best by Asolectin; the least effective reactivation was given by phospholipids from yeast at the middle stages of growth. There are not great differences between the various phospholipids and there is no correlation with unsaturation. If we compare the pattern of appearance of respiration during morphogenesis of yeast mitochondria with the pattern of the capability of the phospholipids from cells at different stages of mitochondrial morphogenesis to restore activity of lipid-depleted yeast mitochondria, we find no correlation. The results of this investigation are consistent with the idea that changes in phospholipids and changes in enzyme activities are not linked by a causal relation.     

Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular beta-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [(14)C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 micromol x g dry yeast(-1)). Stimulation by glucose affected the transport V(max) values, with smaller increases in K(m) values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H(+) symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H(+) symports. A short exposure to glucose allows a closer approach to the sugar/H(+) symport capacity of actively metabolizing cells.     

Regulation of phosphatidylinositol:ceramide phosphoinositol transferase in Saccharomyces cerevisiae.

Maximal phosphatidylinositol:ceramide phosphoinositol transferase activity was measured in yeast cells harvested during the exponential phase of growth. The addition of inositol to the growth medium resulted in a twofold increase in IPC synthase activity in cells grown in the presence or absence of exogenous choline. Enzyme activity was not regulated in yeast inositol biosynthesis regulatory mutants by the addition of inositol to the growth medium.     

Nutrient media for plant parasitic nematodes. 3. Carbohydrate investigations on Aphelenchoides sp

Total carbohydrates of Aphelenchoides sp., harvested from 10-week-old cultures, ranged from 2.54 to 4.38% with a mean of 3.20% dry weight. Approximately 86% was glycogen. The presence of free sugars, trehalose and mannose, were determined by thin-layer chromatography. Mannose was synthesized by the nematodes while trehalose was obtained during feeding from the fungal host.     

Changes in content and composition of phenolic acids during growth of xylem cells of Scots pine

The content and composition of alcohol soluble phenolic acids (PhAs) were studied during cell xylem growth in course of wood annual increment formation in the trunks of Scots pine. Cells of the cambium zone, two stages of expansion growth, and outset of secondary thickening zone (before lignification) within the period of formation of early wood xylem were subsequently isolated from trunk segments of 25-year-old trees with constant anatomical and histochemical control. The amount of free and bound forms of phenolic acids extracted from tissues by 80% ethanol, as well as their ethers and esters, were calculated both per dry weight and per cells. The substantial alteration in content, proportion of fractions and composition of acids has been found between the cambium zone and the outset of secondary thickening of tracheids, and the character of variation depended on the calculation method. The amount of free and bound PhAs and esters and especially ethers calculated per cell had increased at the first stage of extension growth, reduced at the second, and increased in the outset of secondary wall deposition. The pool of bound acids was more than acids by 2–5 times depending on the stage of development of the cells. Sinapic and ferulic acids dominate among free hydroxycinnamic acids. The composition and the content of hydroxycinnamic acids in esters and ethers also depended on the stage of development of the cells. p-Coumaric and sinapic acids were the main aglycons in ethers in the cambium and sinapic and caffeic acids were in the other stages. The esters from cambium included mostly p-coumaric acid and those at other stages of development were sinapic and ferulic acids. The esters included benzoic acid at the first stages of growth. The pool of these esters decreased from the first phase of growth until the outset of cell wall thickening. The level of free benzoic acid increased respectively.     

Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae

The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes.

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Incorporation and accumulation of docosahexaenoic acid from the medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25 degrees C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K2HPO4, which caused browning of the medium, promoted the accumulation of DHA in cells.     

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

THE INFLUENCE OF CULTURAL CONDITIONS ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Apogamous sporophytes formed on Pteridium gametophytes in response to concentrations of certain sugars which supported gametophytic growth. High osmotic concentration of the medium inhibited apogamy, while variations in the basic medium were not stimulatory. Agar, autoclaving, the ammonium ion, and dry media were not required for apogamy. Renewing the medium during an experiment enhanced the apogamous response. Changing the medium at set intervals facilitated the separation of apogamous plant development into gametophytic, initiative, and developmental phases, thus enabling testing of various factors at each of these stages. Apogamy was light-initiated, while the actual development of apogamous sporophytes was caused by light, succinic acid or sugar.     

Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids

Buono, F. (Syracuse University, Syracuse, N.Y.), R. Testa, and D. G. Lundgren. Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids. J. Bacteriol. 91:2291-2299. 1966.-Growth and sporulation were studied in Bacillus cereus by use of an active culture technique and a synthetic medium. A high level of glutamic acid (70 mm) was required for optimal growth and glucose oxidation followed by sporulation even though relatively little glutamic acid was consumed (14 mm). Optimal growth occurred with a combination of 14 mm glutamic acid and 56 mm (NH(4))(2)SO(4), aspartic acid, or alanine. Ornithine or arginine at 70 mm could replace glutamic acid in the synthetic medium without affecting the normal growth cycle. Glutamic acid was not replaced by any other amino acid, by (NH(4))(2)SO(4), or by a combination of either alpha-ketoglutarate or pyruvate plus (NH(4))(2)SO(4). Enzyme assays of cell-free extracts prepared from cells harvested at different times were used to study the metabolism of glutamic acid. Glutamic-oxaloacetic and glutamic-pyruvate transaminases were completely activated (or derepressed) during early stages of sporulation (period of 6 to 8 hr). Alanine dehydrogenase responded in a similar manner, but the levels of this enzyme were much higher throughout the culture cycle. Neither glutamic dehydrogenase nor alpha-ketoglutarate dehydrogenase was detected. Sporulation in a replacement salts medium was studied with cells harvested at different times from the synthetic medium. Cultures 2 to 6 hr old were unable to sporulate in the replacement salts medium unless glutamic acid (7.0 mm) was present. By the 6th hr, cells were in the early stages of sporulation, showing spore septa development. Cultures 8 hr old sporulated in the replacement salts medium. Other metabolic intermediates able to replace glutamic acid in the replacement salts medium were alanine, aspartic acid, and glutamine at equimolar concentrations. Also, ammonium ions in combination with pyruvic, oxaloacetic, alpha-ketoglutaric, or fumaric acid replaced glutamic acid. The likely role of these metabolites is discussed.     

Incorporation and Accumulation of Docosahexaenoic Acid from the Medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25°C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K₂HPO₄, which caused browning of the medium, promoted the accumulation of DHA in cells.     

Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Growth of mammalian cells on substrates coated with cellular microexudates. I. Effect on cell growth at low population densities

Mammalian and avian cells cultured on glass or plastic substrates produce microexudates of cellular macromolecules which remain bound to the substrate when the cells are detached. The gross macromolecular composition of microexudates from a range of diploid, heteroploid, and virus-transformed cells was determined with cells labeled with radioisotopes. Significant differences in the amounts of cellular glycoproteins, proteins, and RNA present in microexudates were found between different cell types and between cells of the same type at different stages of growth. Inoculation of cells onto substrates "coated" with microexudates altered their growth behavior. Microexudates from exponentially growing subconfluent homotypic and heterotypic cell populations enhanced the growth of mouse and chick embryo cells seeded at very low densities, but similar microexudates had no effect on the proliferation of cells seeded at higher densities. The enhanced growth of low-density cell populations seeded on microexudates was compared with the growth enhancement produced by feeder cell layers and conditioned medium.     

Flow cytometric analysis of granulosa cells from developing rat follicles.

Immature rats were treated with diethylstilboestrol (DES) or pregnant mares' serum gonadotropin (PMSG) and forward angle light-scatter (FALS) and 90 degrees light-scatter (90 degrees LS) signals were used to measure the size and the granularity (internal organization) of the granulosa cells, respectively. The results confirmed the presence of two major populations of granulosa cells in the ovaries of both groups of rats, with the same percentage of larger cells in both treatments (52.3% in DES, 49.5% in PMSG). Since DES treatment brings about granulosa cell growth while PMSG treatment causes growth and differentiation, it is evident that there is heterogeneity in granulosa cell sizes during different states of growth and differentiation. There was also heterogeneity in sizes of granulosa cells harvested from follicles of small (less than 210 microns), medium (210-420 microns) and large (greater than 420 microns) diameter. Quadrant analysis of granulosa cells in various fractions collected from Percoll gradients suggested an increase in granularity in the small and large granulosa cell populations. Cell cycle analysis of small and large granulosa cell populations collected from large follicles of rats treated with PMSG indicated that each population was distributed in G0/G1, S and G2/M phases. These results demonstrate that populations of small and large granulosa cells exist in rat ovarian follicles during various stages of growth and differentiation.