Saccharomyces pastorianus immobilized on brewer's spent grain in continuous system for lead ion biosorption

This study investigates the potential application of Saccharomyces pastorianus cells immobilized on brewer's spent grain (BSG) for the removal of lead ions in continuous mode. The effect of initial metal ion concentration on biosorption capacity and the possibility of regeneration and reuse are also assessed. Immobilization of yeast cells on BSG is found to enhance the sorption of lead ions, but only on the first run. Regenerated by washing with 0.05M HCl solution, the biosorbant loses a significant amount of its initial capacity. Degeneration is more severe with higher influent metal concentrations. As a result, the lead ion uptake by yeast immobilized on BSG is not superior to that of BSG alone. These findings reveal that, despite its many potential advantages, yeast immobilized on BSG cannot be recommended for the industrial removal of heavy metal ions from aquatic solutions.     

Effect of a brewer's yeast extract on growth and glucose metabolism of cells in culture.

Brewer's yeast preparations influence glucose metabolism in vivo and in isolated tissues. We have studied the effect of a brewer's yeast extract on glucose metabolism and grwoth of rat hepatoma and human embryonic cells. Growth of the rat hepatoma cells was very much stimulated by the extract in a concentration-dependent manner. Glucose uptake was, on the other hand, appreciably inhibited, and lactate uptake completely abolished by the extract. Insulin stimulated cell growth and inhibited lactate uptake but did not affect the glucose level. Insulin and the extract had additive effects on growth and lactate uptake of the hepatoma cells. The inhibition by the brewer's yeast extract of glucose uptake was, however, antagonized by insulin. Niacin or Cr³⁺, which are suggested to be components of a “glucose tolerance factor” of brewer's yeast, did not affect growth or glucose and lactate uptake. The glucose uptake of the human embryonic cells was strongly inhibited by the brewer's yeast extract. Cell growth and lactate production were not influenced by the extract or by insulin; however, when both insulin and extract were present simultaneously, a slight stimulation of growth and inhibition of lactate production was observed. The results indicate that brewer's yeast can have appreciable direct effects on cells and that not all of these effects are “insulin-like”.     

Uptake and long-distance transport of phosphate,potassium and chloride in relation to internal ion concentrations in barley: evidence of non-allosteric regulation

The extent to which uptake and transport of either phosphate, potassium or chloride are controlled by the concentration of these ions within the root, perhaps through an allosteric mechanism, was investigated with young barley plants in nutrient solution culture. Plants were grown with their roots divided between two containers, such that a single seminal root was continuously supplied with all the required nutrient ions, while the remaining four or five seminal roots were either supplied with the same solution (controls) or, temporarily, a solution lacking a particular nutrient ion (nutrient-deficient treatment). Compared with controls, there was a marked stimulation of uptake and transport of labelled ions by the single root following 24 h or more of nutrient dificiency to the remainder of the root system. This stimulation, which comprised an increased transport to the shoot and, for all ions except Cl^-, increased transport to the remainder of the root system, took place without appreciable change in the concentration of particular ions within the single root. However, nutrient deficiency quickly caused a lower concentration of ions in the shoot and the remaining roots. The results are discussed in relation to various mechanisms, proposed in the literature, by which the coordination of ion uptake and transport may be maintained within the plant. We suggest that under our conditions any putative allosteric control of uptake and transport by root cortical cells was masked by an alternative mechanism, in which ion influx appears to be regulated by ion efflux to the xylem, perhaps controlled by the concentration of particular ions recycled in the phloem to the root from the shoot.     

The use of flocculating brewer's yeast for Cr(III) and Pb(II) removal from residual wastewaters

The use of inexpensive biosorbents to sequester heavy metals from aqueous solutions, is one of the most promising technologies being developed to remove these toxic contaminants from wastewaters. Considering this challenge, the viability of Cr(III) and Pb(II) removal from aqueous solutions using a flocculating brewer's yeast residual biomass from a Portuguese brewing industry was studied. The influence of physicochemical factors such as medium pH, biomass concentration and the presence of a co-ion was characterised. Metal uptake kinetics and equilibrium were also analysed, considering different incubation temperatures. For both metals, uptake increased with medium pH, being maximal at 5.0. Optimal biomass concentration for the biosorption process was determined to be 4.5?g dry weight/l. In chromium and lead mixture solutions, competition for yeast binding sites was observed between the two metals, this competition being pH dependent. Yeast biomass showed higher selectivity and uptake capacity to lead. Chromium uptake kinetic was characterised as having a rapid initial step, followed by a slower one. Langmuir model describes well chromium uptake equilibrium. Lead uptake kinetics suggested the presence of mechanisms other than biosorption, possibly including its precipitation.     

Improved Fermentation Performance of a Lager Yeast after Repair of Its AGT1 Maltose and Maltotriose Transporter Genes

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer''s worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer''s yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. α-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer''s ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous α-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24° Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.The main fermentable sugars in brewer''s wort are maltose (ca. 60% of the total), maltotriose (ca. 25%), and glucose (ca. 15%). In traditional brewery fermentations, worts of about 11° Plato (°P) are used, corresponding to a total fermentable sugar concentration of about 80 g · liter⁻¹. Many modern breweries ferment high-gravity worts (15 to 17°P), and there are efforts to raise the concentration to 25°P, corresponding to a total sugar concentration of about 200 g · liter⁻¹. Industrial use of such very-high-gravity (VHG) worts is attractive because it offers increased production capacity from the same-size brew house and fermentation facilities, decreased energy consumption, and decreased labor, cleaning, and effluent costs (34, 35).Whereas glucose, which is used first, is transported into yeast cells by facilitated diffusion, the α-glucosides maltose and maltotriose are carried by proton symporters (2, 26, 39). Maltose transport seems to have a high level of control over the fermentation rate. Thus, during the early and middle stages of fermentation of brewer''s wort by a lager yeast, the specific rate of maltose consumption was the same as the specific zero-trans maltose uptake rate measured off line with each day''s yeast in each day''s wort spiked with [¹⁴C]maltose (27). Furthermore, introducing a constitutive MAL61 (maltose transporter) gene into a brewer''s yeast on a multicopy plasmid accelerated the fermentation of high-gravity worts (17). Maltotriose is the last sugar to be used in brewing fermentations, and significant amounts of residual maltotriose sometimes remain in beer, causing economic losses (lower yield of ethanol on wort carbohydrate) and possibly undesirable organoleptic effects. The problem of residual sugars in beer is more serious when high-gravity and VHG worts are used. Some, but not all, maltose transporters can also carry maltotriose. The MALx1 genes (x = 1 to 4 and 6) encode transporters that carry maltose efficiently but are generally believed to have little or no activity toward maltotriose (1, 3, 13, 30), although substantial activity toward maltotriose was reported by Day et al. (4). Some yeast strains contain a gene 57% identical to MAL11 that is usually known as AGT1 but is recorded in the Saccharomyces Genome Database (SGDB) as MAL11. The Agt1 transporter has relatively high activity toward maltotriose, as well as maltose (13), and similar K_m values (4 to 5 mM) for these two substrates (4). Alves et al. (1) found that the specific deletion of AGT1 from several Saccharomyces cerevisiae strains also containing at least one MALx1 gene (MAL21, MAL31, and/or MAL41) abolished their ability to transport maltotriose but did not decrease their maltose transport activity. These results supported the belief that the Mal21, Mal31, and Mal41 transporters cannot carry maltotriose, though it remains possible that there are differences between Malx1 transporters from different strains. The same group has also shown (33) that overexpression of AGT1 on a multicopy plasmid in an industrial yeast strain with a very limited ability to ferment maltotriose provided the strain with increased maltotriose uptake activity and the ability to ferment maltotriose efficiently. In 2005, a novel kind of α-glucoside transporter was independently found by two groups (6, 30) in some industrial strains of brewer''s, baker''s, and distiller''s yeasts. These transporters are coded by MTT1 (also called MTY1) genes, which are 90 and 54% identical to the MAL31 and AGT1 genes, respectively. The Mtt1 transporters have high activity toward maltotriose and are the only known α-glucoside transporters with lower K_m values for maltotriose than for maltose (30).Before the discovery of the MTT1 genes, Vidgren et al. (36) sequenced AGT1 genes from two apparently unrelated lager strains and two apparently unrelated ale strains of brewer''s yeast. Surprisingly, at that time (because other maltotriose transporters were not known), the AGT1 genes from the lager strains contained an insertion of one nucleotide, resulting in a premature stop codon, and encoded a truncated, nonfunctional 394-amino-acid polypeptide, whereas those from the ale strains encoded full-length 616-amino-acid transporters. This premature stop codon was later shown (37) to be present in AGT1 genes from all eight of the lager strains tested but was not in any of the four ale strains tested, whereas MTT1 genes were present in all of the lager strains tested but in none of the ale strains tested.In the present work, we have tested whether lager fermentations can be accelerated and residual maltotriose levels decreased by repairing the defective AGT1 genes of lager strains with appropriate DNA sequences from ale strains. Furthermore, the MALx1 and AGT1 genes are repressed by glucose and induced by α-glucosides (9, 16, 19, 25), so that replacing the native AGT1 promoter with a constitutive S. cerevisiae promoter might also increase α-glucoside transport activity and accelerate wort fermentations. The objectives of the present work were to confirm that α-glucoside transport has a high level of control over the rate and extent of wort fermentation and to create a genetically modified lager yeast strain that has improved fermentation performance but contains only Saccharomyces DNA.     

Cooperative regulation by ammonium and ammonium derivatives of nitrite uptake in Chlamydomonas reinhardtii

The role of ammonium in the regulation of nitrite uptake in Chlamydomonas reinhardtii has been investigated under conditions that prevented ammonium assimilation. Prolonged carbon-starvation or inhibition of glutamine synthesis with l-methionine-dl-sulfoximine partially relieved ammonium inhibition of nitrite uptake. However, nitrite uptake was inhibited in both methionine sulfoximine-treated and carbon-starved cells preincubated with ammonium, the inhibition extent in the two cases being directly dependent on the ammonium concentration in the preincubation media. Methionine sulfoximine treatment caused an increase of intracellular ammonium levels. When methionine sulfoximine-treated cells were transferred to ammonium media there existed a linear correlation between intracellular and extracellular ammonium concentration. Addition of methionine sulfoximine to cells with their nitrite uptake system inhibited by ammonium counteracted the effect of ammonium and restored nitrite uptake rate. These results strongly suggest that ammonium itself and a (some) product(s) of its metabolism must act together to block completely nitrite uptake by C. reinhardtii cells. Partial inhibition of nitrite uptake by methylammonium, a structural analogue of ammonium incapable of being used for cell nutrition, supports the above conclusion.     

A1 toxicity in yeast. A role for Mg?

We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae. The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions. Alterations to the PO4, Ca, or K concentration had little effect on toxicity. Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species. The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast. Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F. Fuhrmann, A. Rothstein [1968] Biochim Biophys Acta 163: 325-330). Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg. In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain. Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport. We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Effect of aluminium on the mineral nutrition of rice

A study of the effect of increasing aluminium concentration in a dilute nutrient solution on various aspects of the mineral nutrient uptake by the rice plant has shown that aluminium exerts a stimulation on dry matter production and nutrient uptake until a concentration threshold was reached. The value of this threshold was influenced by nutrient solution composition and cultivar. Its location could be calculated by adjusting to the experimental points a rate law from enzyme kinetics on substrate inhibition curve. On the other hand, total uptake of aluminium and its concentration in the tops was a monotonic function of aluminium concentration in the nutrient solution, the effect of which was greatly enhanced by increased phosphate concentration. A sensitive cultivar accumulated more aluminium than a resistant one.The effect of phosphate on the alleviation of aluminium toxicity was slight in the range of concentration studied.Nitrogen uptake either as ammonium or nitrate nitrogen was clearly influenced by aluminium concentration when its instantaneous value was measured by the technique of the continuously flowing culture solution. The ammonium uptake rate of two cultivars different in sensitivity to aluminium was such that the sensitive variety took up less ammonium and acidified less the culture solution flowing through the root sysstem with a residence time of a few hours.Minor elements concentration in the tops of the rice plants did not seem to be greatly influenced by aluminium with the notable exception of manganese, the uptake of which was clearly depressed by increasing aluminium concentration.Attempts were made at using the speciation of the nutrient solutions with or without aluminium complexation by fluoride in order to rank the various ionic forms of aluminium according to their toxicity. It seems that the well-known result of primary toxicity due to the free Al-ion is also true for rice but that some toxicity is associated with the AlSO₄-ion.     

Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or dl-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption.These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the K dp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.     

Excretion of proline bySaccharomyces cerevisiae during fermentation of arginine-supplemented high gravity wheat mash

Summary The rate of ethanolic fermentation of high gravity wheat mashes bySaccharomyces cerevisiae was increased by nitrogen sources such as ammonium sulfate or arginine. This stimulation was mediated through increased proliferation of cells. Large quantities of proline, however, were excreted by the yeast into the medium when arginine was added as a nutrient supplement. The amount of proline excreted was proportional to the concentration of arginine supplied. Nitrogen sources such as ammonium sulfate or lysine enhanced the production of proline from arginine and its excretion into the medium. Results show that the stimulation of very high gravity fermentation by arginine is not merely through provision of a source of nitrogen but also because it serves as a precursor for the production of proline, a compound which may play a significant role in alleviating the effects of osmotic stress.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.     

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.     

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.     

Nutrient balance in medfly, Ceratitis capitata, larval diets affects the ability of the developing insect to incorporate lipid and protein reserves

The Mediterranean fruit fly [Ceratitis capitata Wiedemann (Diptera: Tephritidae)], or medfly, is mass produced in many facilities throughout the world to supply sterile flies for sterile insect technique programs. Production of sterile males requires large amounts of larval and adult diets. Larval diets comprise the largest economic burdens in the mass production of sterile flies, and are one of the main areas where production costs could be reduced without affecting quality and efficacy. The present study investigated the effect of manipulating diet constituents on larval development and performance. Medfly larvae were reared on diets differing in the proportions of brewer's yeast and sucrose. We studied the effect of such diets on the ability of pupating larvae to accumulate protein and lipids, and on other developmental indicators. Except for diets with a very low proportion of brewer's yeast (e.g., 4%), pupation and adult emergence rates were in general high and satisfactory. The ability of pupating larvae to accumulate lipid reserves and proteins was significantly affected by the sucrose and yeast in the diet, and by the proportion of protein to carbohydrates (P/C). In contrast to previous nutritional studies conducted with other insects, low P/C in medfly larval diets (with excess dietary carbohydrates) resulted in pupating medfly larvae having a relatively reduced load of lipids; medfly larvae protein contents in these diets were, as expected, relatively low. Similarly, high P/C ratios in the diet produced larvae with high protein and lipid contents. Differences with other insects may be due to differential post‐ingestion regulation where a high dietary carbohydrate diet reduces the lipogenic activity of the larvae, and induces a shift from lipid to glucose oxidation. Larvae reared on low P/C diets spent more time foraging in the diet than larvae maintained on a high P/C diet, suggesting a compensatory mechanism to complement nutrient intake. The results suggest that the content of brewer's yeast, the most expensive diet component, could be fine‐tuned without apparently affecting fly quality.     

Effect of some quaternary benzylammonium salts on physiology of yeast

In order to determine the biological activity of eight compounds belonging to a group of quaternary ammonium salts, their influence on the active methionine transport, the integrity of cell membranes, respiration, and viability of Saccharomyces cerevisiae and some other yeast species has been investigated. The earliest effect observed during ammonium salts action on yeast cells is an immediate methionine transport abolishment followed by its fast leakage, which indicates increasing cell membrane degradation. Gradual decline of other biological functions such as respiration and viability is thus a result of disintegration and lack of tightness of the cell membranes. The studied compounds are characterized by a rather unspecific spectrum of action on yeast resulting in irreversible damage of cell walls and cell membranes, which in consequence leads to cell death.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.