Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass

Fermentations with yeastSaccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl₃ into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl₃ into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr³⁺ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr³⁺ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30–45 (μg/g_d.m. of cells. Yeast biomass enriched with chromium ions was extracted with 01 mol/l NH₄OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO₂ production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr³⁺ ions were bonded to the protein molecule.     

Growth efficiency and the specific rate of the yeast Hansenula polymorpha during continuous cultivation on methanol

The growth of Hansenula polymorpha DL-1 in the chemostat (under methanol limitation) and turbidostat was measured. Cultivation with different specific rates of growth mu made it possible to determine the maximum yield of biomass Ys(max)=0.425 and the level of expendables required to maintain Ms=0.023 hr-1. The following parameters describing mu as a function of the concentration of methanol S in the fermenter were found: muo=0.154 hr-1 (maximum growth rate), Ks=1.31 mg/l, Ki=5.35 g/l. The paper emphasizes a very low value of the saturation constant Ks derived from the above experiments and reviews the literature data on the kinetic characteristics of various methanol-grown yeast.     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

Yeast metallothionein. Sequence and metal-binding properties

The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein.     

Cu^2＋对大蒜生长的影响及大蒜根，叶及蒜瓣对Cu^2＋的累积

研究不同浓度（10^-6-10^-4mol/L)硫酸铜（CuSO4,5H2O)溶液对大蒜（Allium satiuwn L.)根,叶和蒜瓣生影响及其这些器官对Cu^2 的积累能力,研究结果指出：在106-5-10^-4mol/L,Cu的处理下,Cu严重影响大蒜根和叶生长,大蒜具有较强吸收和积累Cu^2 的能力,随着Cu^2 处理浓度的增加,大蒜根中的Cu^2 含量递增,大蒜经10^-4mol/L,Cu处理,根部积累了大量的Cu,其含量是对照的52倍,在10^-5和10^-6mol/L Cu处理中,根中Cu的含量分别是对照的13倍和1．4倍,Cu主要积累在极中（10^-5-10^-4mol/L Cu处理）,只有少量的转移到叶子和蒜瓣中。     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+ 胁迫的生长响应与阈限浓度

采用砂培法,研究了匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+胁迫的生长响应及阈限浓度,结果表明:种子萌发率随着4种重金属浓度的增加而下降。对株高的影响是当重金属浓度小于100mg/L时会促进株高生长,高于100mg/L则产生抑制作用。Cu2+显著抑制根系生长,并随浓度的增加抑制效应愈加显著;在Cu2+浓度为600mg/L时匍茎翦股颖的根长比对照下降了93.75%。Cu2+、Zn2+、Pb2+浓度小于200mg/L时会促进地上生物量的增加,但高于200mg/L时,地上生物量会随着3种重金属的增加而减少。Cu2+、Zn2+浓度小于100mg/L或Cd2+、Pb2+浓度小于200mg/L会增加叶绿素的含量,高浓度会降低叶绿素的含量;Cd2+在浓度为600mg/L时显著降低叶绿素含量,与对照相比,下降了43.55%。匍茎翦股颖生长的综合效应分析表明,匍茎翦股颖对Cu2+胁迫最敏感,具有较低的阈限浓度,而Zn2+胁迫对匍茎翦股颖的生长影响最小,阈限浓度相对较高。     

Growth characteristics of bakers' yeast in ethanol

The influence of temperature (15 degrees -40 degrees C) and pH (2.5-6.0) on the continuous growth of bakers' yeast (Saccharomyces cerevisiae) at steady state in 1% ethanol was investigated. Optimal temperature and pH were 30 degrees C and 4.5, respectively. The short-term effect of ethanol concentration (0.1-10.0%) on the yeast growth was assessed in batch culture. Up to 1% of ethanol, the yeast growth increased in function of the ethanol concentration in the medium. The biomass reached a maximum within the interval of 1-4% of ethanol (7.9 and 31.6 g/L, respectively) and decreased at higher concentrations. The residual ethanol concentration in the medium increased rapidly when the initial ethanol concentration exceeded 4%. The best-fit model obtained for growth inhibition as a function of ethanol concentrations was that of Tseng and Wayman: mu(m)S/)K + S( - i (S - S(theta)). With this model, the specific growth rate (mu) decreased linearly as the ethanol concentration increased between the threshold value (S(theta)) of 11.26 g/L to be fully inhibited at 70.00 g/L (S;) an inhibition constant (i) of 0.0048 g L(-1) h(-1), a maximum specific growth rate (mu(m)) of 0.284 h(-1), and a saturation constant (K) of 0.611 g/L were obtained.     

Trehalose metabolism in Saccharomyces cerevisiae during alcoholic fermentation

Strains of Saccharomyces cerevisiae accumulated intracellular trehalose up to 105 mg/g cell dry wt with 90% survival. Viability could be correlated to trehalose levels during ethanol fermentation albeit the disaccharide did not seem to contribute to fermentation yields. Trehalose-6-phosphate synthase showed high activity (up to 279 mu/mg protein) even at high residual sucrose concentration (115 g/l) in the wort suggesting to be a response of yeast cells to the osmotic stress conditions.     

Alteration of cell-wall composition ofFusarium oxysporum by copper stress

A strain ofFusarium oxysporum tolerated copper in the growth medium at concentrations up to 600 mg/L. The optimum growth was obtained at 200 mg Cu/L. The mycelium acquired a blue color in the presence of copper. The copper content of isolated cell walls obtained from mycelium grown in the presence of 600 mg Cu/L was 1.5 times higher than that of cell walls obtained from mycelium grown at 200 mg Cu/L and it contained 2.2 and 3.3% copper at 200 and 600 mg Cu/L, respectively. The amount of protein and total sugars increased in both the mycelium and its isolated cell walls in the presence of copper in the growth medium, chitin was also increased in the cell wall, reaching its maximum amount at 200 mg Cu/L— about 2.4 times higher than without copper. Most of amino acid concentrations in the cell wall were increased in the presence of 200 mg Cu/L and decreased above this concentration. Isoleucine, leucine, tyrosine, phenylalanine, and arginine showed the highest increase at this concentration. The altered cell walls obtained from mycelium grown at 200 and 400 mg Cu/L could rebind individual metals more than the control cell walls could. Rebinding of individual metals was in the order Zn>Fe>Ni>Cu>Co. Rebinding of copper by isolated cell walls depended on pH and temperature.     

Sensitivity of Embryos and Larvae of the Sea Urchin Strongylocentrotus intermedius to Effects of Copper Ions

The effect of copper ions in seawater (0.02 mg/l) on the early stages of development of the sea urchin Strongylocentrotus intermedius was studied. Copper exposure from fertilization or the prism stage retarded development and growth and led to abnormalities in the morphology of the embryos and larvae. However, if development to the pluteus stage proceeded in clean seawater, an increased copper concentration did not inhibit the growth of larvae. If sea urchin embryos at fertilization and the prism stage were maintained for 1–2 days in seawater containing 0.02 mg Cu/l and then transferred to clean seawater, the adverse consequences of this exposure remained present after 48 h.     

Effect of Chronic Fenvalerate Intoxication on Tissue Concentration of Copper in Goats and Further Exploration of Its Mechanism

This experiment was designed to assess the effect of chronic fenvalerate toxicity on tissue Cu concentration in goats and to explore the pathways responsible for it. A significant decrease in tissue Cu concentration of kidney, heart, and brain while an increase in the liver were recorded in fenvalerate intoxicated goats at 15 mg/kg b.w. orally daily for 270 days. Concentration of total Cu, protein-free Cu, and protein-bound Cu in the wet intestine of fenvalerate-treated goats revealed a significant decrease in Cu concentration of the intestine due to the decrease in trichloroacetic acid (TCA)-insoluble Cu, while TCA-soluble Cu remained almost unaffected. Rabbit duodenal loop technique was used to assess the relative absorption of nonisotopic copper in a living animal. This technique enabled to compare Cu absorption from the lumen of three closely associated loops, each receiving 100 µg of copper along with different doses (0, 15, and 30 µg) of fenvalerate. A significant dose-dependent decrease in Cu absorption from the lumen due to fenvalerate treatment was recorded. A decrease in total copper (TCA-insoluble fraction) suggested an interference in active transport of copper due to the inhibition of absorption of protein-bound copper. It was concluded that fenvalerate interfered in copper absorption mostly by inhibiting its active or mediated transport.     

Effects of small increases in copper levels on culturable basidiomycetous yeasts in low‐nutrient soils

Aims: Investigating the effect of perturbations, with relatively low Cu concentrations, on yeast community composition in low‐nutrient virgin soil. Methods and Results: Culturable soil yeast populations were monitored at an experimental site treated with the fungicide copper oxychloride (10 mg Cu per kg soil). Yeast numbers were unaffected by additional Cu; however, a shift in yeast community composition from Hymenomycetes to Urediniomycetes species occurred. Subsequent growth experiments conducted with a synthetic liquid medium revealed that hymenomycetous and urediniomycetous yeasts were affected differently by 1 and 10 mg l^?1 Cu. Soil microcosm experiments then indicated that additional 10 mg kg^?1 Cu may improve the competitive ability of urediniomycetous yeasts in the presence of hymenomycetous yeasts. Conclusions: The shift from hymenomycetous to urediniomycetous yeasts, as a result of slightly increased soil Cu levels, may be because of hymenomycetous yeasts being more sensitive to elevated Cu levels and urediniomycetous yeasts having an improved competitive ability in the presence of elevated Cu levels. Significance and Impact of the Study: Yeast community composition of pristine low‐nutrient soils may change as a result of perturbations with relatively low concentrations of Cu. Urediniomycetous yeasts should be studied as potential bio‐indicators of Cu perturbations.     

Influence of 2,4-dinitrophenol on the characteristics of activated sludge in batch reactors

The response of activated sludge characteristics to the presence of 2,4-dinitrophenol (dNP) in batch cultures was investigated in this study. The sludge yield slightly decreased with an increase in dNP concentration. At 10 mg l(-1), or lower, dNP significantly reduced sludge yield and relative specific growth rates (mu/mu0), but didn't substantially affect its relative specific chemical oxygen demand removal rate (q/q0). Presence of dNP at 1-20 mg l(-1) increased the specific oxygen uptake rate of activated sludge, and slightly changed its hydrophobicity. An analysis on inhibition indicated that the reduction in sludge yield in the presence of dNP was mainly attributed to the significant decreased sludge growth, rather than the reduced substrate degradation.     

The effect that nickel,copper, and zinc salts have on seed germination and initial ontogenesis of water parsnip (<Emphasis Type="Italic">Sium latifolium</Emphasis> L.) and wood club-rush (<Emphasis Type="Italic">Scirpus silvaticus</Emphasis> L.)

The effect that nickel, copper, and zinc sulfates have on seed germination and the initial stages of the ontogenesis of water parsnip and wood club-rush has been investigated. Nickel and copper in the concentration range of 250–500 mg/l and zinc at a concentration of 500 mg/l were the most toxic for water parsnip seeds, while, for the wood club-rush seeds, maximum toxicity was observed at Ni and Cu concentrations ranging from 50 to 500 mg/l and at Zn concentrations of 250–500 mg/l. The development of water parsnip seedlings was normal at Ni concentrations of 1–25 mg/l, Cu concentrations of 1–10 mg/l, and Zn concentrations up to 50 mg/l; the development of wood club-rush seedlings was normal at a Ni concentration of 1 mg/l, and Cu and Zn concentrations of 1–25 mg/l. A further increase in the concentration caused photosynthesis suppression, slower growth of the vegetation organs, and their subsequent necrosis. Water parsnip is more resistant to the toxicants.     

The effects of copper on the microbial community of a coral reef sponge

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators for sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha-proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 microg l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 microg l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 microg l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction in the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 microg l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morpho-type actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 microg l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 microg l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 microg l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 microg l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.     

Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger

The effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration. The optimum pH values for growth and metal ion accumulation were determined as 5.0, 4.5 and 3.5 for copper(II), lead(II) and chromium(VI) ions, respectively. Although all metal ion concentrations caused an inhibition effect on the growth of A. niger, it was capable of removing of copper(II) and lead(II) with a maximum specific uptake capacity of 15.6 and 34.4 mg g⁻¹ at 100 mg dm⁻³ initial copper(II) and lead(II) concentration, respectively. Growth of A. niger was highly effected by chromium(VI) ions and inhibited by 75 mg dm⁻³ initial chromium(VI) concentration since some inhibition occurred at lower concentrations.     

Effect of copper and manganese on the de novo generation of protease-resistant prion protein in yeast cells

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to bind manganese and copper, the latter being involved in the normal function of the protein. Indeed, upon expression in yeast we noted a major increase in intracellular copper and a decrease in manganese. Interestingly, protease-resistant PrP(Sc)-like protein (PrP(res)) formation was induced when PrP-expressing yeast cells were grown in copper- and/or manganese-supplemented media. The pattern of PrP banding in SDS-PAGE was dominantly determined by manganese. This conformational transition was stable against EDTA treatment but not in the presence of the copper chelators bathocuproinedisulfonic acid or clioquinol. Conclusively, PrP itself influences manganese and copper metabolism, and a replacement of copper in PrP complexes with manganese is highly likely under the condition of copper depletion or if excess amounts of copper and manganese are present. Taken together, our present study demonstrates the involvement of PrP in the regulation of intracellular metal ion homeostasis and uncovers copper and, more severely, manganese ions as in vivo risk factors for the conversion into PrP(Sc).     

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts.

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.     

Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei (Boone) held in 35 per thousand seawater were challenged with Vibrio alginolyticus at a dose of 3 x 10(5) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l(-1). Mortality of shrimp in 5, 10 and 20 mg l(-1) Cu2+ was significantly higher than those in 1 mg l(-1) Cu2+ and the control solution after 24-96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l(-1) Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l(-1) or greater for 24h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l(-1) caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l(-1) or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l(-1) Cu2+ was considered to be cytotoxic to the host.