Washed Yeast Cells Research Articles

BIOLOGICAL CONTROL OF LASIODIPLODIA ROT OF MANGO FRUITS BY YEASTS

Ninety nine yeast isolates were isolated from surface of mango fruits and were tested in vivo in preliminary study for biocontrol potential of Lasiodiplodia rot of mango fruits. According to primary screening, nine isolates were selected to continue the secondary screening using different concentrations of the washed yeast cells in water suspension to study their biocontrol efficacy at 16±1°C. It was found that using washed cells of yeast isolate Mg 147 (Candida multisgemmis) at 1x109, 5x108 and 2x108 CFU/ml produced complete protection for 14 days to wounds of mango fruit inoculated with spore suspension of the fungusLasiodiplodia theobromae 1x105 conidia/ml. Meantime, no lesion developed on the mango fruit treated with the isolates Mg 39 (Pichia guilliermondii strainA) at the highest tested colony forming unit (CFU) levels, i.e. 1x109 and 5x108 CFU/ml. Supernatant of the yeast cultures, tested in secondary screening, did not prevent spore germination of L. theobromae or decay of wounded mango fruit but had inhibitory effect. However, 1-Methylcyclopropene (1-MCP) treatment significantly reduced the infected area mm2 on mango fruit inoculated with L. theobromae (1x105 conidia/ml) during the 14 days of storage at 16±1°C and improved efficacy of isolates Mg147 and Mg39. When applied as combined treatments between 1-MCP and biocontrol agents, no lesion developed on the fruit treated with the isolate Mg 147 at 1x 108 CFU/ml or higher. Meanwhile, no lesion developed on the fruit treated with the isolate Mg 39 at 2x 108 CFU/ml or higher while, the percentages of rot reduction were ranged between 94.1% - 81.3% for concentrations 1x108 and 6.6x107 CFU/ml respectively.

Sample introduction of single selenized yeast cells (Saccharomyces cerevisiae) by micro droplet generation into an ICP-sector field mass spectrometer for label-free detection of trace elements

We have applied a micro droplet generator (μDG) for sample introduction of single selenized yeast cells into a sector field ICP-MS, which was operated in a fast scanning mode with sampling rates of up to 10 kHz, to measure single cells time resolved with 100 μs integration time. Selenized yeast cells have been used as a model system for preliminary investigation. The single cells to be measured have been embedded into droplets and it will be shown that the time duration of a single cell event always is about 400 to 500 μs, and thus comparable to the time duration of a droplet without a cell. A fixed droplet generation rate of 50 Hz produced equidistant signals in time of each droplet event and was advantageous to separate contribution from background and blank from the analytical signal. Open vessel digestion and a multielement analysis were performed with washed yeast cells and absolute amounts per single cell were determined for Na (0.91 fg), Mg (9.4 fg), Fe (5.9 fg), Cu (0.54 fg), Zn (1.2 fg) and Se (72 fg). Signal intensities from single cells have been measured for the elements Cu, Zn and Se, and histograms were calculated for about 1000 cell events. The mean elemental sensitivities measured here range from 0.7 counts per ag (Se) to 10 counts per ag (Zn) with RSD's from 49% (Zn) to 69% (Se) for about 1000 cell events.

Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses

C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.

Biosynthesis of Crystalline Silver and Gold Nanoparticles by Extremophilic Yeasts

The biosynthesis of Ag and Au nanoparticles (NPs) was investigated using an extremophilic yeast strain isolated from acid mine drainage in Portugal. Three distinct studies were performed, namely, the growth of yeast strain in presence of metal ions, the use of yeast biomass for the metal nanoparticles synthesis, and of the supernatant obtained after 24-hour incubation of yeast biomass in water. The extremophilic strain under study was able to grow up to an Ag ion concentration of 1.5 mM whereas an increase of Au ion concentration over 0.09 mM caused a strong inhibitory effect. A successful route for the metal NPs synthesis was obtained using the yeast biomass. When the washed yeast cells were in contact with Ag or Au solutions, AgNPs smaller than 20 nm were produced, as for the AuNPs diameter ranged from 30 to 100 nm, as determined through transmission electron microscopy and confirmed by energy-dispersive X-ray spectra. The supernatant-based strategy provided evidence that proteins were released to the medium by the yeasts, which could be responsible for the formation and stabilisation of the Ag NPs, although the involvement of the cell wall seems fundamental for AuNPs synthesis.

A vibrating membrane bioreactor (VMBR): Macromolecular transmission—influence of extracellular polymeric substances

The vibrating membrane bioreactor (VMBR) system facilitates the possibility of conducting a separation of macromolecules (BSA) from larger biological components (yeast cells) with a relatively high and stable macromolecular transmission at sub-critical flux. This is not possible to achieve for a static non-vibrating membrane module. A BSA transmission of 74% has been measured in the separation of 4 g/L BSA from 8 g/L dry weight yeast cells in suspension at sub-critical flux ( 20 L / ( m 2 h ) ) . However, this transmission is lower than the 85% BSA transmission measured for at pure 4 g/L BSA solution. This can be ascribed to the presence of extracellular polymeric substances (EPS) from the yeast cells. The initial fouling rate for constant sub-critical flux filtration of unwashed yeast cells is 3–4 times larger than for washed yeast cells ( 18 ( mbar / h ) / 5 ( mbar / h ) ) . At sub-critical flux, an EPS transmission of around 32% is measured for a pure yeast cell suspension. Thus, EPS and BSA are “competing” in being transmitted which might explain the lowered BSA transmission in the presence of yeast cells. Additionally, EPS heavily foul the membranes, leading to a 86% permeability drop and a fouling resistance 6 times larger than the membrane resistance after 5 1 2 h of constant sub-critical flux filtration ( 20 L / ( m 2 h ) ) of pure 8 g/L dry weight yeast cell suspensions. Thus, the addition of hydraulic resistance caused by EPS might also explain the lowered BSA transmission, in the presence of yeast cells, since the membrane pores might be narrowed or partly blocked. EPS is, furthermore, able to cause a relatively large permeability drop even on a membrane module pre-fouled by EPS.

Crossflow filtration of washed and unwashed yeast suspensions at constant shear under nominally sub-critical conditions

Operation at sub-critical fluxes can be used to control membrane fouling. The original definition of the critical flux stated that operation was sub-critical if no or negligible fouling occurred. Over time there has been a relaxing of the criteria and many now consider a low rather than zero rate of fouling to be indicative of sub-critical operation especially when dealing with complex feeds. Here the region of low fouling is termed “nominally sub-critical”. Unwashed yeast, washed yeast and extra polymeric substance (EPS) suspensions were filtered at controlled fluxes to investigate the role of cells and soluble components in nominally sub-critical conditions using ultrafiltration (UF) and microfiltration (MF) membranes. As the UF membrane could not be effectively cleaned it was not used in the later part of the study. Tracking of membrane resistance, R m * of the 0.2 μm membranes was continued through the whole study. After the initial increase, R m * rises very slowly, increasing on average only 0.4% after each experiment and cleaning cycle. For the MF membranes, the rate of fouling increased with increasing feed concentration, increasing membrane pore size and decreasing shear stress. The effect of increasing shear stress was to reduce the amount of reversible fouling but the irreversible component was invariant with shear stress for the range studied. Also the rate and reversibility of fouling were found to be sensitive to changes in pH. The sum of the rates of transmembrane pressure (TMP) rise for washed yeast cells and EPS suspensions were in all cases found to be lower than that for unwashed yeast. The origin of the additional resistance is discussed and other relevant literature reviewed.

Effects of organic and inorganic additives on flotation recovery of washed cells of Saccharomyces cerevisiae resuspended in water

Separation of microbial cells by flotation recovery is usually carried out in industrial reactors or wastewater treatment systems, which contain a complex mixture of microbial nutrients and excretion products. In the present study, the separation of yeast cells by flotation recovery was carried out using a simple flotation recovery systems containing washed yeast cells resuspended in water in order to elucidate the effects of additives (defined amounts of organic and inorganic acids, ethanol, surfactants and sodium chloride) on the cellular interactions at interfaces (cell/aqueous phase and cell/air bubble). When sodium chloride, organic acids (notably propionic, succinic and acetic acids) and organic surfactants (sodium dodecyl sulphate (SDS), cetyltrimethylammonium bromide (CTAB) and Nonidet P40) were added to the flotation recovery system, significant increases in the cell recovery of yeast hydrophobic cells ( Saccharomyces cerevisiae, strain FLT-01) were observed. The association of ethanol to acetic acid solution (a minor by-product of alcoholic fermentation) in the flotation recovery system, containing washed cells of strain FLT-01 resuspended in water, leading to an increased flotation recovery at pH 5.5. Thus, the association among products of the cellular metabolism (e.g., ethanol and acetic acid) can improve yeast cell recovery by flotation recovery.

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×109 colony-forming units per milliliter or higher and pathogen spore suspensions of 5×104 spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.

Effects of Cryptococcus laurentii (Kufferath) Skinner on biocontrol of postharvest decay of arbutus berries

Cryptococcus laurentii was tested as a biocontrol agent for reducing natural decay of arbutus berries caused by Penicillium citrinum and Verticicladiella abielina in semi-commercial postharvest trials. Three different preparations of C. laurentii were compared for antagonistic efficiency. Washed C. laurentii cells provided better protection against decay than yeast cultured in broth without washing while the culture supernatant free of yeast cells provided no protection. The protection provided by the washed yeast cells was dose-dependent. Cryptococcus laurentii was also effective in controlling decay at low temperature (4°C). The efficacy of C. laurentii was enhanced by the addition of 2% CaCl2. Agar disks of C. laurentii NYDA cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. citrinum or V. abielina. Spore germination of the pathogens in potato dextrose broth was strongly inhibited in the presence of active cell suspensions.

Direct observation of membrane cleaning via rapid backpulsing

Direct visual observation (DVO) was used to view membrane surfaces at different times in crossflow microfiltration of yeast suspensions with rapid backpulsing at varied backpulsing duration and pressure. The DVO photos show that the membranes are more effectively cleaned by longer backpulse durations and higher backpulse pressures. However, a tradeoff exists, as longer and stronger backpulses also have greater permeate loss during each backpulse. The net flux was increased two-fold and four-fold with backpulsing under optimum conditions for 0.2 μm Anopore and cellulose acetate membranes, respectively, fouled by 0.05 g/L washed yeast cells. Shorter, stronger backpulses resulted in higher net fluxes than did weaker, longer backpulses after 2 h of filtration.

Rapid Colorimetric Assay and Yeast Surface Display for Screening of Highly Functional Fungal Lignin Peroxidase.

Rapid screening method using color reaction was developed to quantitatively evaluate the lignin peroxidase (LiP) activity. For 2,4-dichlorophenol (DCP) degradation activity, this method correlated well with the HPLC method. The lignin peroxidase H2 gene was isolated from Phanerochaete chrysosporium, a white-rot fungus. The molecular weight of the recombinant LiP was ca. 38 kDa, which corresponded to the predicted mature LiP H2 gene. We performed cloning into pYD vector for yeast cell surface display of the gene. The surface expression was checked by the rapid, colorimetric screening method. The supernatant did not change the color, but the washed yeast cells changed the color from white to red, which confirmed the enzymes were located on the cell surface. Five colonies expressing the LiP H2 gene at high levels were selected. The average amount of 2,4-DCP degradation was about 23%. It was approximately 63% degradation level of the LiP from the wild type fungus. This screening and selection protocol is being applied to the DNA shuffling effort to screen LiPs with improved functionality and stability.

Expulsion of uracil and thymine from the yeast Saccharomyces cerevisiae: contrasting responses to changes in the proton electrochemical gradient.

The outflow of uracil from the yeast Saccharomyces cerevisiae is known to be relatively fast in certain circumstances, to be retarded by proton conductors and to occur in strains lacking a uracil proton symport. In the present work, it was shown that uracil exit from washed yeast cells is an active process, creating a uracil gradient of the order of -80 mV relative to the surrounding medium. Glucose accelerated uracil exit, while retarding its entry. DNP or sodium azide each lowered the gradient to about -30 mV, simultaneously increasing the rate of uracil entry. They also lowered cellular ATP content. Manipulation of the external ionic conditions governing delta mu H+ at the plasma membrane had no detectable effect on uracil transport in yeast preparations thoroughly depleted of ATP. It was concluded that uracil exit is probably not driven by the proton gradient but may utilize ATP directly. It is known that thymine is not normally absorbed by yeast. However, thymine expulsion was here observed during deamination of the substrate 5-methylcytosine in the presence of glucose. In the absence of glucose, or following ATP depletion, thymine uptake from the medium only occurred when delta mu H+ was dissipated, either by DNP or azide, or by manipulation of the external ionic environment. The yeast expelled absorbed thymine when delta mu H+ was restored to the physiological range. The properties of the system corresponded to those of an H+/thymine antiport that is distinct from the mechanism expelling uracil.

The putative electrogenic nitrate-proton symport of the yeast Candida utilis. Comparison with the systems absorbing glucose or lactate.

Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization.

Evaluation of antibody coating of yeasts in urine as an indicator of the site of urinary tract infection

Antibody coating of yeasts (Candida sp. and Torulopsis sp.) found in urine specimens was investigated to ascertain whether the presence of such coating might identify the site of urinary tract infection. Washed yeast cells obtained by centrifugation of fresh urine specimens were reacted with fluorescein-conjugated goat antihuman immunoglobulins (Ig) G, A, and M and examined by fluorescent microscopy. IgG was found on the surface of all species of yeast encountered in all urine specimens evaluted, whereas there was variability of IgA AND IgM coating. Antibody coating with IgG, IgA, AND IgM was also demonstrated on yeasts from other body sites (sputum, gastrostomy, oral, etc.). Control experiments confirmed the specificity of the reactions. Thus, it appears that yeasts from any body site are coated with antibodies. These results are in contrast to recent work with bacteria which showed that the presence of antibody-coated (IgG) bacteria indicates upper urinary tract infection (pyelonephritis) while bacteria are not coated with antibodies in lower urinary tract infection. Since all yeasts from all body sites tested were found to be coated with antibody regardless of the clinical situation, the presence of surface antibody has no diagnostic value in identifying the site of urinary tract infection with yeasts.

Microbial production of D-mannitol and D-fructose from glycerol.

AbstractIn view of the recent development that some petrochemical products are efficiently available as substrates for the fermentation industry, glycerol manufactured from propylene by chemical synthesis would also be hoped for the purpose. This paper describes some of the factors influencing mannitol production from glycerol by Torulopsis yeasts and a microbial conversion of glycerol to D‐fructose via mannitol, in which two sequential steps of yeast and Acetobacter fermentation are involved. Torulopsis mannitofaciens CBS 5981 and Torulopsis vcrsatilis CBS 1752, exceptionally good mannitol producers, were selected for the study. High concentrations of nitrogen sources and KH2PO4 in the medium markedly decreased mannitol yield in spite of good utilization of the substrate. T. mannitofaciens produced mannitol in yield of 31% of the glycerol consumed at optimal condition. The fermentation by washed yeast cells gave much higher mannitol yield of more than 50%. A sequential fermentation process was carried out without isolation and purification of the intermediate and yielded.51.7%. D‐fructose from the glycerol.

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of alpha-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released alpha-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

It was confirmed that washed yeast cells produced isoamyl alcohol and isobutanol from either pyruvic acid or α-ketoisovaleric acid. At the same time α-ketoisocaproic acid, a presumed intermediate to isoamyl alcohol, was found. These results seem to support the presumptive scheme that pyruvic acid converts to α-ketoisocaproic acid via acetolactic acid and α-keto,isovaleric acid, from which isoamyl alcohol and isobutanol are formed.

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

It was found that washed yeast cells produced active amyl alcohol from aspartic acid, homoserine or α-hydroxy-aceto-n-butyric acid each of which was considered the intermediate in the synthetic pathway of active amyl alcohol and isoleucine. At the same time n-butanol and α-keto-β-methyl-n-valeric acid were detected in the fermented solutions, and α-keto-n-butyric acid was formed in the fermented solutions contained the former two compounds. These results seemed to support the reliability of this pathway. Acetylpropionyl and 2,3-pentanediol were formed from α-hydroxy-aceto-n-butyric acid.

The Formation of Higher Alcohols in the Fermentaion of Amino Acids by Yeast

1. It was confirmed that washed yeast cells produced active amyl alcohol and n-butanol from oxalacetic acid, aspartic acid, homoserine of HAB. At the same time α-keto-β-methyl-n valeric acid was formed. 2. These results strengthen the reliability of the presumptive scheme of the formation of active amyl alcohol proposed by us. 3. Acetylpropionyl and 2, 3-pentanediol were formed from HAB.

Aerobic fermentation and the depletion of the amino acid pool in yeast cells.

The amino acid pool of yeast cells, Saccharomyces cerevisiae, incubated with galactose remains at a constant level for 100 minutes. This is 30 minutes beyond the time at which the oxidative phase of the induced-enzyme formation begins. Washed yeast cells, the pools of which have been depleted 60 per cent by incubation with glucose, do not replenish their pools as do washed cells incubated without a substrate. These facts indicate that the induced enzymes are formed at least partially from pool-replenishing amino acids. The time of onset of pool depletion is the time at which the aerobic fermentation phase of induced-enzyme formation begins for cells incubated with galactose. With 0.1 per cent galactose the respiratory phase begins at 100 minutes but no aerobic fermentation nor pool depletion occurs. The rates of respiration and aerobic fermentation are constant for four glucose concentrations from 0.1 to 1.0 per cent. The amount of aerobicfermentation is proportional to the initial concentration of glucose. Amino acid pool depletion occurs for all concentrations but depletion ceases and is followed by pool replenishment after aerobic fermentation is complete. Ultraviolet radiations, which delay the appearance of the respiratory phase of induced-enzyme formation, completely eliminate both the appearance of aerobic fermentation and pool depletion. The results indicate an intimate association between aerobic fermentation and amino acid pool depletion.