Yeast cultivation in lettuce brine

The use of lettuce brine, a by-product of the vegetable fermentation industry, as a medium for yeast cultivation was investigated. Six strains of yeast, Saccharomyces sp., Pichia sp., Rhodotorula sp., Candida sp., Kluyveromyces sp. and Trichospora sp. grew well in diluted lettuce brine under aerobic conditions. The acid brine becomes neutral after yeast cultivation. The yeast strains reached the maximum growth after the first day of cultivation. Trichosporon sp. was found to grow best in the brine with the maximum specific growth rate at 0.09 h⁻¹ and growth yield of 67%. This revised version was published online in November 2006 with corrections to the Cover Date.     

Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Did Gause Have a Yeast Infection?

We planned to develop predator–prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator–prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia‐yeast time‐series data, from Gause. We hypothesised that if the model simulated predator–prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self‐sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator–prey dynamics.     

Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Ammonia production from yeast extract and its effect on growth of the hyperthermophilic archaeonSulfolobus solfataricus

Utilization of yeast extract and formation of byproduct metabolite were investigated for hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). In both batch and fed-batch cultivations ofS. solfataricus, maximal cell density, NH₄ ⁺ ion production and pH change were highly dependent on the ratio of yeast extract to glucose in the medium. Variation of NH₄ ⁺ ion level was identified as a major cause of pH change during cultivation, and acidification of culture broth was attributed to consumption of NH₄ ⁺ ions rather than formation of acid byproducts. It was also observed that increase of NH₄ ⁺ ion concentrations in the medium resulted in greater degree of growth inhibition.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

Diethylpyrocarbonate—An effective agent for the sterilization of different types of nutrient media

A simple and convenient method has been tested for the steriltzation of nutrient media for long-term cultivation of plant cells. Diethylpyrocarbonate is suitable for this task in concentrations about 1000 mg l^-1 The cells cultivated for 15 subsequent passages on media treated by DPC had the same growth parameters, production pattern and ability to transform exogeneous organic compounds as did the controls. The method is suitable for the preparatian of both liquid and agar media, for stabilization of stock solutions and for sterilization of cultivation vessels and tubing.Abbreviations DPC diethylpyrocarbonate - medium MS nutrient medium according to Murashige and Skoog - NAA naphtaleneacetic acid     

Effect of malic acid on the growth kinetics of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis>

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h^–1. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h^–1. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol^–1 hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg^–1 cell h^–1 in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Subcellular Effects of Cytochalasin B and Dimethylsulfoxide on Paramecium aurelia*

SYNOPSIS. Paramecium aurelia syngen 4, stock 57 (sensitive) cultivated in Cerophyl infusion were exposed to cytochalasin B CB and dimethylsulfoxide (DMSO), the solvent for CB, to distinguish between the effects of these agents on a cellular system. DMSO significantly inhibited survival, fission rate, [³H]leucine incorporation, and cell size. CB-treated cells generally had slower division and poorer survival rates than cells exposed to the equivalent DMSO concentration, although the [3H]leucine incorporation was generally greater at the lower CB concentrations than for DMSO alone. As seen by electron microscopy and a new grycerination technic for observing polysomes, DMSO caused nuclear (nucleolar, chromatin) abnormalities as well as membrane degradation and polysomal breakdown; CB caused the formation of aberrant membrane structures and ribosomal tetramers, crystals, and tubes.     

Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis

The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase^® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L^?1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD₆₀₀) of approximately 40 AU (corresponding to over 60 g L^?1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.     

Purification by silica gel chromatography using dialysis tubing and characterization of sophorolipids produced from <Emphasis Type="Italic">Candida bombicola</Emphasis> grown on glucose and arachidonic acid

The yeast Candida bombicola (ATCC 22214) grown on primary carbon source glucose (100 g l⁻¹) and secondary carbon, arachidonic acid (2 g l⁻¹) produced mixture of sophorolipids up to 1.44 g l⁻¹. The crude product was a heterogeneous mixture of sophorolipids, which are glycolipids of sophorose linked to the fatty acid through glycosidic bond between ω and ω−1 carbon of arachidonic acid. The derived sophorolipids were isolated by silica gel chromatography using dialysis tubing. The purified sophorolipids were characterized by ESI-MS and FT-IR. Acid hydrolysis of the resolved sophorolipids were characterized by ESI-MS for the presence of 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (20-HETE) and 19-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (19-HETE), compounds of pronounced pharmaceutical importance.     

Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans

Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4 g l^–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5 g l^–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16 g l^–1 h^–1 were obtained when the C/N ratios in the feed were 10, 20 and 100 g octanoic acid g^–1 ammonium nitrate, respectively.     

Einfluß von Kupfer-, Mangan- und Zinkionen auf die makromolekulare Zusammensetzung der Hefe Candida utilis M 72

The ions of Cu²⁺, Zn²⁺ and Mn²⁺ affect significantly the macromolecular composition of yeast Candida uiilis M 72. Crude protein and nucleic acid content increase and carbohydrate content decreases with rising intracellular ion concentration. Lipid fraction is influenced by metallic ions only little. The correlation between intracellular ion concentration and cell composition of macromolecules can be described by simple equations.     

Production,Purification and Properties of Ribonuclease from Aspergillus niger

Aspergillus niger NRC–A–1–233 was cultivated by the shaking method. The optimal cultural conditions for ribonuclease (RNase) production were: composition of medium: sucrose, 15%; NH₄NO₃, 0.2%; KH₂PO₄, 0.1%; MgSO₄·7 aq., 0.025%; initial pH, 2.2; shaking conditions: 50 ml of medium /500 ml flask; cultivation time, 120 hr. The RNase was purified by acid clay treatment and chromatography on DEAE-cellulose and Sephadex G–75 columns. The purified RNase was homogeneous by ultracentrifuge and disc electrophoresis.

The molecular weight of the RNase was estimated to be 28,500 on SDS-polyacrylamide gel and its isoelectric point was 2.8 by Ampholine electrofocusing method. Digestion rate of RNA by the RNase was 100%. The RNase did not have an exact base specificity and produced four kinds of 3′-nucleotides from yeast RNA.     

The rhythm of mating in Paramecium aurelia, syngen 3

The periodic mating behavior of some stocks of Paramecium aurelia, syngen 3, exhibits features typical of circadian rhythms. In the most extensively studied stock (37^p), rhythmicity persists at least four days in continuous darkness, but disappears rapidly in continuous illumination (200 foot-candles). The period of the free-running rhythm is 22.2 hours, and relatively insensitive to ambient temperature. Cycles of illumination and temperature can regulate the mating rhythm. Changes in illumination at specified times in the circadian cycle will induce shifts in the phase of the rhythm. Stock differences with respect to the persistence of the rhythm and the environmental control of its phase have been observed.     

Aerobic Respiration of Kappa Particles from Paramecium aurelia,Stock 51*

SYNOPSIS. Kappa particles from killer cultures of stock 51 Paramecium aurelia were purified and their respiration measured polarographically. The slight bacterial contaminations in the kappa preparations were not significant. Freshly collected kappa in dilute buffer at room temperature had an endogenous Q_O2 of 17.0 ± 1.6 μl/mg dry weight/hr (mean ± standard error). The Q_O2 decayed 50% in 5 hr. Among the sugars tested only glucose and sucrose increased the respiratory rate of kappa. The di- and tri-carboxylates of the tricarboxylic acid cycle stimulated the respiration of kappa. KCN, CO and 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO) inhibited respiration. These findings ensure an organismic status for kappa and justify the belief that it is bacterial in origin.