Some properties of the adenosine triphosphatase systems of two yeast species,saccharomyces cerevisiae and rhodotorula glutinis

1. Total ATPase levels were determined in homogenate fractions of baker's yeast, Saccharomyces cerevisiae K and Rhodotorula glutinis. The maximum ATPase activities in 8000 X g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pih-1 (gDS)-1, respectively; the activities in the sediment were somewhat higher. Exponential cells of S. cerevisiae K and R. glutinis exhibited higher ATPase levels than did the stationary cells. 2. The total ATPase activity in both yeast species showed a maximum at ph 6.8 a minimum at pH 7.2, and another broader masimum around pH 8.0. 3. No significant NaK-ATPase activity was detected in baker's yeast, in either the exponential or the stationary cells of R. glutinis, and in exponential S. cerevisiae K cells in the pH range of 6.0-9.3. 4. Stationary cells of S. cerevisiae K exhibited, at pH 7.0-8.5, A Na,K-ATPase activity attaining 9% of total ATPase level. 5.3 X 10(-3) M phenylmethyl sulphonyl fluoride had no effect on the total ATPase level in S. cerevisiae and inhibited the activity in R. glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence. 6. 1.5 M urea lowered the ATPase activity in R. glutinis by 68% but had no effect on S. cerevisiae cells. 10(-5) M dicyclohexylcarbodiimide suppressed the ATPase activity in S. cerevisiae and R. glutinis by 74 and 79%, respectively. Neither agent revealed and additional Na,K-ATPase activity. 7. The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lower flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions. The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.     

Seasonal occurrence of yeasts and yeast-like organisms in the river Danube

One hundred and seventy yeast strains belonging to 14 genera and 29 species were isolated from 112 water samples of the river Danube in the area of Bratislava. The samples were collected through the year from April to March.Saccharomyces cerevisiae, Candida maltosa, Aureobasidium pullulans, Cystofilobasidium capitatum, Rhodotorula glutinis, Geotrichum candidum, and Candida krusei were the most frequent. The basidiomycetous yeasts and yeast-like organisms with oxidative metabolism were present in approximately equal numbers to those with fermentative metabolism. Saccharomyces cerevisiae was the dominant yeast and was isolated from 50% of all samples examined and represented approximately one quarter of the yeast community.Yeast densities ranged from 100 to 21,100 CFU per litre. The highest population density was observed in October. Cryptococcus albidus, Saccharomyces cerevisiae, Rhodotorula glutinis, and Aureobasidium pullulans formed the main part of the yeast population in this month.     

Streptococcus thermophilus and its biosurfactants inhibit adhesion by Candida spp. on silicone rubber.

The adhesion of yeasts, two Candida albicans and two Candida tropicalis strains isolated from naturally colonized voice prostheses, to silicone rubber with and without a salivary conditioning film in the absence and presence of adhering Streptococcus thermophilus B, a biosurfactant-releasing dairy isolate, was studied. Coverage of 1 to 4% of the surface of silicone rubber substrata with adhering S. thermophilus B gave significant reductions in the initial yeast adhesion regardless of the presence of a conditioning film. Mechanistically, this interference in yeast adhesion by S. thermophilus B was not due to direct physical effects but to biosurfactant release by the adhering bacteria, because experiments with S. thermophilus B cells that had released their biosurfactants prior to adhesion to silicone rubber and competition with yeasts did not show interference with initial yeast adhesion. The amounts of biosurfactants released were highest for mid-exponential- and early-stationary-phase bacteria (37 mg.g of cells-1 [dry weight]), but biosurfactants released by stationary-phase bacteria (14 mg.g of cells-1 [dry weight]) were the most surface active. The crude biosurfactants released were mixtures of various components, with a glycolipid-like component being the most surface active. A lipid-enriched biosurfactant fraction reduced the surface tension of an aqueous solution to about 35 mJ.m-2 at a concentration of only 0.5 mg.ml-1. The amount of biosurfactant released per S. thermophilus B cell was estimated to be sufficient to cover approximately 12 times the area of the cross section of the bacterium, making biosurfactant release a powerful defense weapon in the postadhesion competition of the bacterium with microorganisms such as yeasts. Preadsorption of biosurfactants to the silicone rubber prior to allowing yeasts to adhere was as effective against C. albicans GB 1/2 adhesion as covering 1 to 2% of the silicone rubber surface with adhering S. thermophilus B, but a preadsorbed biosurfactant layer was less effective against C. tropicalis GB 9/9.     

Comparison of enzymatic antioxidant defence systems in different metabolic types of yeasts

The enzymatic defence system in the 2 yeasts Kluyveromyces marxianus and Rhodotorula glutinis, differing in their mode of oxygen uptake and energy generation, was characterized and compared with the well-studied facultatively fermentative Crabtree-positive Saccharomyces cerevisiae strain. Twofold higher superoxide dismutase (SOD) and catalase activities were detected in K. marxianus and R. glutinis when cells were cultured on glucose. Further increases of 10%-15% in SOD activity and 30%-50% in catalase were measured in all studied yeasts strains after transfer to media containing ethanol. An evaluation of the ratio of Cu/Zn SOD / Mn SOD was performed as a measure of the oxidative metabolism. A 20% decrease was observed when the respiratory source of energy was ethanol, with the lowest ratio being observed for the oxidative type of K. marxianus yeasts. Electrophoretic analysis revealed that all tested strains possess active Cu/Zn and Mn SODs. A reverse electrophoretic mobility pattern of K. marxianus and R. glutinis SOD enzymes was observed in comparison with the same couple in S. cerevisiae. The investigation of electrophoretic profile of catalase enzymes showed that alongside their different taxonomic status and fermentative capacity, all tested strains possess 2 separate catalases. The role of antioxidant enzymes in preventing oxidant-induced cytotoxicity (treatment with hydrogen peroxide, paraquat, and menadione) was shown.     

High-productivity fermentation process for cultivating industrial microorganisms

Summary High-productivity continuous fermentation processes have been developed for the production of important industrial microorganisms in specially designed fermentors.Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces fragilis, andCandida utilis yeasts have been grown in bench-scale fermentors at cell densities of over 120 g/l, whileEscherichia coli, Bacillus megaterium, Methylomonas sp. andPseudomonas putida bacteria have been cultivated to cell densities of more than 110 g/l. Productivities (g cells per 1 per h) greater than 25 have been achieved in both bench-scale and 1500-liter fermentors with yeasts, and values as high as 55 have been achieved with bacteria in the bench-scale fermentor. The microorganisms were grown on defined media using ammonia for pH control and as nitrogen source. The fermentor, capable of high oxygen and heat transfer rates, was operated at constant volume with continuous feed and product discharge. The high-productivity process reduces fermentor size, media sterilization requirements, and may under some circumstances eliminate waste and recycle streams. It can also be applied to a variety of biological products.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病(Penicilium expansum Link)的丝孢酵母(Trichosporon pullulans (Lindner.) Diddens and Lodder)、罗伦隐球酵母(Cryptococcus laurentii (Kuffer.) Skinner)和粘红酵母(Rhodotorula glutinis (Fresen.) F. C. Harrison).其中,抑病效果最好的T. pullulans 是一种用于采后果实生物防治的新型拮抗菌.研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果.实验结果表明:酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C. laurentii的细胞悬浮液中加入0.18 mol/L 的CaCl2能显著提高其抑病能力,但增加CaCl2 对T. pullulans 和R. glutinis 的抑病效果却没有明显作用.     

Sterol content, fatty acid composition of phospholipids, and permeability of labeled ethylene glycols in relation to salt-tolerance of yeasts

Two yeasts, the salt-tolerant Debaryomyces hansenii and the non-tolerant Saccharomyces cerevisiae were grown in basal media (4 m M NaCl) and also a high salinities that produced a similar salt stress in the two species in terms of growth rate reduction (i.e., 1.4 M NaCl for S. cerevisae and 2.7 M NaCl for D. hansenii ). A study was made of the sterol content, the fatty acid composition of the phospholipids, and the permeation of a series of tritiated ethylene glycols of graded molecular weights. On the basis of cell dry weight the amount of total and free sterols increased in both species when cultured at high salinity. Irrespective of growth medium salinity, the molar ratio of free sterols to phospholipids was higher in D. hansenii than in S. cerevisiae . Increased salinity produced only minor changes in the fatty acid composition of the phospholipids in D. hansenii , whereas in S. cerevisiae there was a marked decrease of linolenic acid with a concomitant increase of linoleic acid.
In both yeasts there was an energy linked component in the uptake of ethylene glycol, which component could be inhibited by sodium azide and N -ethylmaleimide. The passive permeability for ethylene-, diethylene- and triethylene glycol increased for both species at increased salinity. This increase was more pronounced for S. cerevisiae than for D. hansenii . Polyethylene glycol of M , 200 as well as higher polyethylene glycols appeared to be excluded or very slowly admitted by the yeasts.     

Genetic immobilization of proteins on the yeast cell surface

A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and alpha-type proteins involved in mating. The fusion gene was introduced into S. cerevisiae and expressed under the control of several promoters. Appearance of the fused proteins expressed on the cell surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques. Alpha-galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T cell receptor from mammalian cells have been successfully immobilized on the yeast cell wall in the active form. Recently, surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria. The yeasts were termed 'arming yeasts' with biocatalysts or functional proteins. Such arming cells displaying glucoamylase from Rhizopus oryzae and alpha-amylase from Bacillus stearothermophilus, or carboxymethylcellulase and beta-glucosidase from Aspergillus acleatus, could assimilate starch or cellooligosaccharides as the sole carbon source, although S. cerevisiae cannot intrinsically assimilate these substrates. GFP-arming cells can emit green fluorescence from the cell surface in response to the environmental conditions. The approach described in this review will enable us to endow living cells, including yeast cells, with novel additional abilities and to open new dimensions in the field of biotechnology.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病（Peniclium expansum Link)的丝孢酵母（Trichosporon pullulans(Lindner.)Diddens and Lodder)。罗伦隐球酵母（Cryptococcus laurentii(Kuffer.)skin-ner)和粘红酵母（Rhodotorula glutinis(Fresen.)F.C.Harrison)。其中,抑病效果最好的T.pullulans是一种用于采后果实生物防治的新型拮抗菌,研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果。实验结果表明;酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C.laurentii的细胞悬浮液中加入0.18mol/L的CaCl2能显著提高其抑病能力。但增加CaCl2对T.pullulans和R.glutinis的抑病效果却没有明显作用。     

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture.     

Comparison of glycolytic NADH oscillations in yeasts Saccharomyces cerevisiae and Saccharomyces carlsbergensis

The possible mechanism of synchronization of NADH oscillations in yeasts were studied. It was shown that the synchronization time depends on cell concentration in suspension. Synchronization of oscillations after acetaldehyde addition was found in Saccharomyces carlsbergensis whereas in S. cerevisiae oscillations were synchronized after adding potassium cyanide. It is possible, that synchronization of oscillations in S. cerevisiae requires low concentration of acetaldehyde and the high acetaldehyde concentration synchronizes oscillations in S. carlsbergensis. In addition, a possible mechanism of synchronization by acetaldehyde in proposed.     

Uptake of L-lysine by a double mutant ofSaccharomyces cerevisiae

A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport. Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system. The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol. The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate). The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine. The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule. The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre. It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached. Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.     

Cross-effects of extracellular factors of adaptation to stress in Luteococcus casei and Saccharomyces cerevisiae

Saccharomyces cerevisiae yeasts (lower eukaryotes) were shown to produce a protein exometabolite with reactivation activity. We demonstrated cross-effects of extracellular protein factors of adaptation to stress (heat and UV irradiation) in yeasts and Luteococcus casei bacteria. The possibility for isolation and partial purification of protein exometabolites from the culture liquid of yeasts and bacteria by similar methods, as well as the similarity of elution profiles for the active proteins in high-performance liquid chromatography, suggests that the proteins (or fragments thereot) of the organisms studied are homologous.     

Sulfonate-sulfur assimilation by yeasts resembles that of bacteria

Abstract Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae . Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.     

粘红酵母和酿酒酵母联合处理味精废水

为了解决味精废水中高NH4+浓度抑制油脂微生物的生长和油脂积累问题,采用粘红酵母和酿酒酵母联合处理味精废水的方法:首先利用酿酒酵母降解味精废水(MSG)中NH4+,然后将处理后的废水进一步发酵培养合成油脂。研究结果表明:用经酿酒酵母预处理过的味精废水作为粘红酵母的培养基发酵时,粘红酵母的生物量为33.3 g/L,油脂产率为18.16%,COD降解率为50.6%,NH4+的降解率为93.9%。比粘红酵母单独处理味精废水,NH4+的降解率提高了6.14倍,生物量、油脂产率和COD降解率分别提高了8.1%、30.06%和9.58%。     

利用蔗渣半纤维素水解液产油酵母的筛选、鉴定及发酵实验

对已分离到的产油酵母通过木糖摇瓶发酵,从中筛选到2株H2-1和J2-2利用蔗渣半纤维素水解液高产油脂酵母,其利用蔗渣半纤维素水解液油脂得率系数分别为13.49和10.28,均显示出对蔗渣半纤维素水解液的高转化率.根据常规生理生化特征和26S rDNA序列分析结果,确认H2-1为小红酵母(Rhodotorula minuta),J2-2为粘红酵母(Rhodotorula glutinis).     

Bacterial-fungal biofilms in flowing water photo-processing tanks

A complex seven species model community, including bacteria and fungi, was selected from organisms isolated from the walls of an industrial flowing water system. Growth rates of the species were determined in single and mixed batch culture growth. The rates were found to be significantly higher in mixed culture for Pseudomonas alcaligenes and Flavobacterium indologenes and higher in single culture for Xanthomonas maltophilia , Rhodotorula glutinis and Fusarium solani , whereas no significant difference was recorded for Alcaligenes denitrificans and Fusarium oxysporum . All species attached to PVC in single and mixed culture to form biofilms. Xanthomonas maltophilia , Alc. denitrificans , Ps. alcaligenes and F. solani biofilm cell densities cm^-2 were significantly higher than attachment of the component species in mixed culture. Statistical analyses showed a significant difference in rate of colonization between single and mixed cultures for some species. No significant difference was noted between mixed culture cell densities cm^-2 at laminar flows of Reynolds number 2·7 and 5·4.     

Role of Competition for Sugars by Yeasts in the Biocontrol of Gray Mold of Apple

The yeasts, Cryptococcus laurentii BSR-Y22 or Sporobolomyces roseus FS-43-238, but not Saccharomyces cerevisiae BY-1, reduced gray mold ( Botrytis cinerea ) when applied to wounds of apples (cv. Golden Delicious) which were stored at 22IC for 7 days or 3IC for up to 2 months. The role of competition for sugars by these yeasts as a mechanism of antagonism was investigated at 22IC. Populations of C. laurentii and S. roseus in wounds were 6-9 times greater than those of S. cerevisiae from 1 to 7 days following inoculation. Yeasts in wounds utilized more 14C-labelled fructose, glucose or sucrose than conidia of B. cinerea during 48 h, but yeasts did not differ in their utilization of sugars. Utilization of 14C-sugars by yeasts in vitro was greater at most sampling times for conidia; the uptake after 48 h was always greater for yeasts and the addition of nitrogen did not alter this result. The utilization of 14C-sugars by S. roseus in vitro was greater than that in the other yeasts. The uptake and utilization of 14C-fructose by C. laurentii or S. roseus was greater than that of S. cerevisiae , but the utilization of glucose or sucrose by C. laurentii and S. cerevisiae was similar and the uptake of these sugars by these yeasts did not differ. Yeasts mixed with conidia in sterile, dilute solutions of fructose, glucose or sucrose, or in dilute apple juice inhibited conidial germination compared with no-yeast controls; S. cerevisiae was less effective than C. laurentii or S. roseus . Only yeasts rapidly depleted sugars from juice or sugar solutions. Yeasts did not alter the pH or oxygen content of dilute juice to the detriment of conidial germination. These results strongly suggest that competition for sugars by yeasts played a role in the biocontrol of gray mold, but that some other factor(s) most likely contributed to differences in efficacy between the yeasts.     

Influence of oxygen on the biosynthesis of cellular fatty acids,sterols and phospholipids during alcoholic fermentation by Saccharomyces cerevisiae and Torulaspora delbrueckii

Saccharomyces cerevisiae and Torulaspora delbrueckii were grown under different O₂ availabilities on grape must. Oxygen requirements for the two yeasts were different: under anaerobic conditions, S. cerevisiae produced a higher percentage of unsaturated fatty acids, and had a greater cell yield and fermentation activity than T. delbrueckii. Addition of ergosterol (25mg/l) and oleic acid (31mg/l) caused total recovery of cellular growth and the fermentation activity of S. cerevisiae in anaerobiosis, but not of T. delbrueckii. However a short period of aeration to a 48 h culture in anaerobiosis, led to total recovery of the cellular growth and fermentation activity in both yeasts. Likewise, the effect of a short aeration period on unsaturated fatty acid biosynthesis was similar for both species.     

Candida albicans and Saccharomyces cerevisiae induce interleukin-8 production from intestinal epithelial-like Caco-2 cells in the presence of butyric acid

Intestinal epithelial cells (IEC) are important in initiation and regulation of immune responses against numerous foreign substances including food, microorganisms and their metabolites in the intestine. Since the responses of IEC against yeasts have not yet been well understood, we investigated the effects of Candida albicans, Saccharomyces cerevisiae, and their cell wall components on interleukin-8 (IL-8) secretion by the IEC-like Caco-2 cells. Live cells of both yeast species stimulated Caco-2 cells to produce IL-8 only in the presence of butyric acid, which is a metabolite produced by intestinal bacteria. S. cerevisiae zymosan and glucan also enhanced IL-8 secretion. Treatment of Caco-2 cells with butyric acid increased the expression of mRNAs coding for Toll-like receptor 1 (TLR1), TLR6 and dectin-1, which recognize zymosan. C. albicans induced more IL-8 secretion and also decreased transepithelial electrical resistance more rapidly than S. cerevisiae. These results suggest that both yeasts in the intestine stimulate the host's mucosal immune systems by interacting with IEC.