Flocculation onset in Saccharomyces cerevisiae: the role of nutrients

AIMS: To examine the role of the nutrients on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. For cells grown in chemically defined medium (yeast nitrogen base with glucose) or in rich medium (containing yeast extract, peptone and fermentable sugars: fructose or maltose), the onset of flocculation occurred after the end of exponential respiro-fermentative phase of growth being coincident with the attainment of the lower level of carbon source in the culture medium. Cells, in exponential respiro-fermentative phase of growth, transferred to a glucose-containing medium without nitrogen source, developed a flocculent phenotype, while these carbon source starved cells, in the presence of all other nutrients that support growth, did not flocculate. In addition, cells in exponential phase of growth, under catabolite repression, when transferred to a medium containing 0.2% (w/v) of fermentable sugar (fructose or maltose) or 2% (v/v) ethanol, showed a rapid triggering of flocculation, while when incubated in 2% (v/v) glycerol did not develop a flocculent phenotype. CONCLUSIONS: The onset of flocculation occurs when a low sugar and/or nitrogen concentration is reached in culture media. The triggering of flocculation is an energetic dependent process influenced by the carbon source metabolism. The presence of external nitrogen source is not necessary for developing a flocculent phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients on the onset of flocculation in NewFlo phenotype yeast strains. This information might be useful to the brewing industry, in the control of yeast flocculation, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality.     

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae

AIMS: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. CONCLUSIONS: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.     

Carbohydrate carbon sources induce loss of flocculation of an ale-brewing yeast strain

AIMS: To identify the nutrients that can trigger the loss of flocculation under growth conditions in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. Yeast growth with metabolizable carbon sources (glucose, fructose, galactose, maltose or sucrose) at 2% (w/v), induced the loss of flocculation in yeast that had previously been allowed to flocculate. The yeast remained flocculent when transferred to a medium containing the required nutrients for yeast growth and a sole nonmetabolizable carbon source (lactose). Transfer of flocculent yeast into a growth medium with ethanol (4% v/v), as the sole carbon source did not induce the loss of flocculation. Even the addition of glucose (2% w/v) or glucose and antimycin A (0.1 mg l(-1)) to this culture did not bring about loss of flocculation. Cycloheximide addition (15 mg l(-1)) to glucose-growing cells stopped flocculation loss. CONCLUSIONS: Carbohydrates were the nutrients responsible for stimulating the loss of flocculation in flocculent yeast cells transferred to growing conditions. The glucose-induced loss of flocculation required de novo protein synthesis. Ethanol prevented glucose-induced loss of flocculation. This protective effect of ethanol was independent of the respiratory function of the yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients in the control of the flocculation cycle in NewFlo phenotype yeast strains.     

Flocculation in Saccharomyces cerevisiae: a review

The present work reviews and critically discusses the aspects that influence yeast flocculation, namely the chemical characteristics of the medium (pH and the presence of bivalent ions), fermentation conditions (oxygen, sugars, growth temperature and ethanol concentration) and the expression of specific genes such as FLO1, Lg‐FLO1, FLO5, FLO8, FLO9 and FLO10. In addition, the metabolic control of loss and onset of flocculation is reviewed and updated. Flocculation has been traditionally used in brewing production as an easy and off‐cost cell‐broth separation process. The advantages of using flocculent yeast strains in the production of other alcoholic beverages (wine, cachaça and sparkling wine), in the production of renewal fuels (bio‐ethanol), in modern biotechnology (production of heterologous proteins) and in environmental applications (bioremediation of heavy metals) are highlighted. Finally, the possibility of aggregation of yeast cells in flocs, as an example of social behaviour (a communitarian strategy for long‐time survival or a means of protection against negative environmental conditions), is discussed.     

Flocculation onset in Saccharomyces cerevisiae: effect of ethanol, heat and osmotic stress

AIMS: To examine the effect of different stress conditions on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]; plasma membrane integrity was accessed using propidium iodide and the staining of the yeast cell wall was performed using calcofluor white M2R. Cells in exponential phase of growth were subjected to different stress conditions. The addition of 1%, 3% and 5% (v/v) ethanol, 1% and 3% (v/v) isopropanol or a brief heat shock (52 degrees C, 5 min), did not induce an early flocculation phenotype when compared with control cells. The addition of 10% (v/v) ethanol, a continuous mild heat-stress (37 degrees C) or an osmotic stress (0.5 or 1 mol l(-1) of NaCl) did not induce a flocculent phenotype. CONCLUSIONS: Flocculation seems not to be induced as a response to different chemical (ethanol and isopropanol) and physical (heat and osmotic) stress conditions. Conversely, osmotic and ethanol [10% (v/v)] stress, as well as a continuous mild heat shock (37 degrees C), have a negative impact on the phenotype expression of flocculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the control of yeast flocculation. This information might be useful to the brewing industry, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality, as well as in other biotechnological industries where flocculation can be used as a cell separation process.     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。     

The use of succinic acid, as a pH buffer, expands the potentialities of utilisation of a chemically defined medium in Saccharomyces cerevisiae flocculation studies

The pH, viability and flocculation during the growth of Saccharomyces cerevisiae NCYC 1214 in rich medium (YEPD) and in chemically defined medium (YNB) were compared. As the pH fell from 5.3 to 2.2, the viability fell to 20% and no flocculation was observed in YNB medium, despite the high flocculation in buffer. With the addition of 50 mM succinic acid and 3.1 mM Ca²⁺, similar growth to YEPD, with 99% of viability and flocculation in culture medium, was obtained.     

Chemical modifications of the cell-surface components of Lactobacillus fermentum FTPT 1405 and their effect on the flocculation of Saccharomyces cerevisiae

The effect of treatment of Lactobacillus fermentum with several protein- and carbohydrate-modifying reagents on the bacterium's ability to flocculate Saccharomyces cerevisiae was investigated. The proteinaceous nature of the cell-surface components of L. fermentum which are responsible for floc formation was confirmed by inactivation of floc formation following photo-irradiation, with Methylene Blue or Rose Bengal as sensitizer, or acylation with acetic anhydride, maleic anhydride or acetylimidazole, and by the reaction of the components with nitrous acid, I₂ and performic acid.The phenolic hydroxyl group of tyrosine and the indole group of tryptophan appear essential for flocculation. Proteinaceous components of the yeast cell surface and carbohydrate components on the bacterial cell surface were not required for flocculation but carbohydrate residues on the yeast surface were essential.     

Induction of flocculation of brewer's yeast strains of Saccharomyces cerevisiae by changing the calcium concentration and pH of culture medium

Strains of Saccharomyces cerevisiae (NCYC 1190, 1214 and 1364) behaved as nonflocculent in defined culture medium, as well as the strain NCYC 1214 in rich medium. Flocculation was induced by Ca²⁺ addition and/or by correcting the pH to a suitable value. Since the free Ca²⁺ concentration is pH-dependent, these two factors (total Ca²⁺ concentration and pH) cannot be dissociated and are the critical parameters governing flocculation, in culture medium, of the brewing strains studied.     

On-line determination of flocculating Saccharomyces cerevisiae concentration and growth rate using a capacitance probe

During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method.     

Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing

We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.     

Enhancement of maltose utilisation by Saccharomyces cerevisiae in medium containing fermentable hexoses

Saccharomyces cerevisiae are unable to maintain high rates of fermentation during transition from catabolism of hexoses to maltose. This phenomenon, termed ‘maltose lag’, presents problems for the baking, brewing and distilling industries, which rely on yeast catabolism of mixtures of hexoses and maltose. Maltose utilisation requires the presence of maltose permease and α-glucosidase (maltase), encoded by MAL genes. Synthesis of these is induced by maltose and repressed by glucose. One strain of baker’s yeast used in this work exhibited a marked maltose lag, whereas a second strain exhibited a shorter lag during conversion from hexose to maltose metabolism. The extent of the lag was linked to the levels of maltose permease and maltase in cells at the time of inoculation into mixed sugar medium. This view is supported by results showing that pulsing yeast with maltose to induce expression of MAL genes prior to inoculation into mixed sugar medium, enhanced sugar fermentation. Maltose pulsing of yeasts could therefore be useful for enhancing some fermentations relevant to baking and other yeast industries. Received 24 December 1988/ Accepted in revised form 18 March 1999     

Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells

Like bacteria, fungi growing in biofilms are often embedded in a so-called extracellular matrix (ECM), a complex and species-specific mixture of compounds secreted by cells in the biofilm. The precise physiological role of this ECM and its importance for the stress and drug resistance that is so characteristic of biofilms remain vague. Here, we describe the discovery of an ECM produced by flocculating Saccharomyces cerevisiae cells. Although S. cerevisiae has long been believed not to produce an ECM, our results indicate that flocculating cells secrete a mixture of glucose and mannose polysaccharides that surrounds flocculating cells. This matrix impedes the penetration of large molecules into the floc, but does not seem to play a role in the resistance of flocculating cultures to drugs and ethanol. Together, our results provide a new model system to study the formation and biological role of microbial extracellular matrices.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Genome-scale patterns in the loss of heterozygosity incidence in Saccharomyces cerevisiae

Former studies have established that loss of heterozygosity can be a key driver of sequence evolution in unicellular eukaryotes and tissues of metazoans. However, little is known about whether the distribution of loss of heterozygosity events is largely random or forms discernible patterns across genomes. To initiate our experiments, we introduced selectable markers to both arms of all chromosomes of the budding yeast. Subsequent extensive assays, repeated over several genetic backgrounds and environments, provided a wealth of information on the genetic and environmental determinants of loss of heterozygosity. Three findings stand out. First, the number of loss of heterozygosity events per unit time was more than 25 times higher for growing than starving cells. Second, loss of heterozygosity was most frequent when regions of homology around a recombination site were identical, about a half-% sequence divergence was sufficient to reduce its incidence. Finally, the density of loss of heterozygosity events was highly dependent on the genome’s physical architecture. It was several-fold higher on short chromosomal arms than on long ones. Comparably large differences were seen within a single arm where regions close to a centromere were visibly less affected than regions close, though usually not strictly adjacent, to a telomere. We suggest that the observed uneven distribution of loss of heterozygosity events could have been caused not only by an uneven density of initial DNA damages. Location-depended differences in the mode of DNA repair, or its effect on fitness, were likely to operate as well.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Use of Saccharomyces cerevisiae for Cu2+ removal from solution: the advantages of using a flocculent strain

A flocculent strain of Saccharomyces cerevisiae S646-1B accumulated more Cu²⁺ (81 nmol mg^–1 dry wt) than the isogenic (except for the marker genes ade1 and trp1 and the gene FLO1) non-flocculent strain S646-8D (30 nmol mg^–1 dry wt), in the first 10 min of contact of the cells with Cu²⁺. Additionally, this strain flocculated in solutions of 0.2 mM Cu²⁺, Ni²⁺, Zn²⁺ and Cd²⁺. The potential of using flocculent strains in the bioremediation of heavy metals contaminated waste waters is discussed.     

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: advantages of using dead biomass

Aim:  The capacities of live and heat-killed cells of Saccharomyces cerevisiae at 45°C for the removal of copper, nickel and zinc from the solution were compared.
Methods and Results:  Kinetic studies have shown a maximum accumulation of Ni²⁺ and Zn²⁺ after 10 min for both types of cells, while for Cu²⁺ this was attained after 30 and 60 min for dead and live cells, respectively. Equilibrium studies have shown that inactivated biomass displayed a greater Zn²⁺ and Ni²⁺ accumulation than live yeasts. For Cu²⁺, live and dead cells showed similar accumulation. Fluorescence, scanning electron microscopy and infrared spectroscopy studies have shown that no appreciable structural or molecular changes occurred in the cells during the killing process. The increased metal uptake observed in dead cells can be most likely explained by the loss of membrane integrity, which allows the exposition of further metal-binding sites present inside the cells.
Conclusions:  Heat-killed cells showed a higher degree of heavy metal removal than live cells, being more suitable for further bioremediation works.
Significance and Impact of the Study:  Dead flocculent cells can be used in a low cost technology for detoxifying metal-bearing effluents as this approach combines an efficient metal removal with the ease of cell separation.     

Extracellular enzyme loss during polyelectrolyte flocculation of cells from fermentation broth

Association of extracellular protein product with flocculated cells reduces product yield. Here, partitioning of the enzyme subtilisin between the liquid and polyelectrolyte-flocculated and sedimented Bacillus increased as the polymer dosage was increased beyond that necessary to obtain optimum floc character (brain floc) for cell removal by centrifugation. Partitioning to the cell floc is partly physical entrapment at all polymer dosages; however, at higher levels there is also direct interaction between the polyelectrolyte and enzyme. Enzyme loss was not likely due to pH denaturation during the flocculation process because conditions were within the stable pH range of the enzyme. The direct interaction between polyelectrolyte and enzyme was characterized through turbidimetric titrations and partitioning studies. Neither changes in the polymer feed concentration nor the method of polymer addition reduced the enzyme loss at dosages optimal for cell removal.