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.

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 degrees 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 Ni(2+) and Zn(2+) after 10 min for both types of cells, while for Cu(2+) this was attained after 30 and 60 min for dead and live cells, respectively. Equilibrium studies have shown that inactivated biomass displayed a greater Zn(2+) and Ni(2+) accumulation than live yeasts. For Cu(2+), 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.

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.

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.

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.

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.

Viability and release of complexing compounds during accumulation of heavy metals by a brewer's yeast.

Saccharomyces cerevisiae NCYC 1190 cells accumulated (after 1 h) lead and cadmium at similar levels, and to a lesser degree also copper. During heavy metal accumulation, there was a considerable loss of viability of copper-treated cells (about 99% in the first 20 min of contact with the metal), and a less pronounced lethal effect on cadmium- and lead-treated cells (about 66% and 46% after 1 h of contact with cadmium or lead, respectively) was detected. During copper accumulation, a leakage of UV-absorbing compounds and inorganic phosphate was observed; this did not occur with lead, whereas with cadmium a small amount of leakage of inorganic phosphate was detected. The filtrates of copper-treated cells contained copper-binding molecules. The copper-binding capacity of the filtrates increased with time according to the release of inorganic phosphate and UV-absorbing compounds. These compounds can bind an appreciable quantity of metal ions, making them unavailable for cell uptake and thus reducing the efficiency of heavy metals removal by yeast cells.

Flocculation onset, growth phase, and genealogical age in Saccharomyces cerevisiae.

Flocculation onset, the time during the fermentative cycle at which the strains of Saccharomyces cerevisiae become flocculent, is an important factor in the brewing industry. The flocculation ability of Flo 1 phenotype (strain NCYC 869) remained practically unchanged throughout the growth and seems to be insensitive to the presence of nutrients of the culture medium. On the contrary, the flocculation of NewFlo phenotype (strain NCYC 1195) exhibited a cyclic behaviour. It was found that the loss of flocculation in the early growth was the result of two combined effects: the dismantling of the flocculation mechanism of the cells coming from the inoculum and the nonflocculent state of the new cells produced after growth has started. The onset of flocculation of strain NCYC 1195 in the cultural conditions used in this work coincided with the end of the exponential growth, when the minimum glucose level in the culture medium was attained. It was demonstrated that it is possible to manipulate the flocculation onset by changing the initial glucose concentration in the culture medium.

Effect of cultural and nutritional conditions on the control of flocculation expression in Saccharomyces cerevisiae.

The effect of cultural (temperature and pH) and nutritional conditions (nitrogen and carbon source) on the flocculation expression of three strains was studied. The strains' flocculation ability was determined by placing the cells in a stationary phase of growth in standard flocculation conditions. The flocculation ability of strain NCYC 1195, recently classified in the literature as the NewFlo phenotype, was more sensitive to growth temperature than Flo1 phenotype strains (NCYC 869 and NRRL Y265). The initial pH of the culture medium did not affect the flocculation ability of Flo1 phenotype strains but in the case of strain NCYC 1195 flocculation was repressed when the initial pH of the culture medium was below 3.5. Flocculation in strain NCYC 1195 was also repressed in defined culture medium; this inhibition was not related to a deficiency in any particular nitrogen source, but rather to the poor buffering capacity of the defined medium. All strains showed strong flocculation when grown in glucose, but were nonflocculent in glycerol. It was clearly demonstrated that the phenotypic expression of flocculation could be induced or repressed by changing cultural and nutritional conditions. Two distinct behaviours were also displayed with regard to the effect of the cultural conditions upon flocculation, namely the effect of pH. These different behaviours can be used to distinguish the two flocculation phenotypes.

Population dynamics of flocculating yeasts.

The problem of understanding the recognition and specific interactions in a population of yeast flocculating cells is discussed. The biochemistry, physiology and genetics of flocculation is briefly reviewed. Yeast flocculation requires the expression of a specific protein (lectin) on flocculent cells, and carbohydrate (receptors) on neighbouring cells. Adhesion experiments performed with cells whose flocculation is repressed by growth conditions, indicating that the inhibition of flocculation is due to inhibition or inactivation of 'lectin-like' component. Additionally, using adhesion experiments, it is demonstrated that cells of non-flocculent strain interact by establishing a true bond with flocculent cells rather than by entrapment inside the floc matrix. As phenotypic expression of flocculation, for several strains, is shown to be repressed, modulated or induced by modifying growth conditions, the constitutiveness and inducibility of flocculation are also discussed.

Interaction between flocculent and nonflocculent cells of Saccharomyces cerevisiae.

Interaction between nonflocculent and flocculent cells of Saccharomyces cerevisiae was studied. Adhesion experiments were done using three types of nonflocculent cells and a flocculent one. Two types of nonflocculent cells were obtained from the flocculent strain by changing environmental growth conditions. The integration of nonflocculent cells in the flocs was observed by two different methods: measurement of the sedimentation capacity of mixtures and microscopic observation of stained nonflocculent cells blended with flocculent cells. It was possible to verify that cell-cell interaction corresponds to a true stable binding and not to a simple entrapment inside the floc matrix.