Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain ofSaccharomyces cerevisiae

Abstract

Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.Flor or velum formation by certain wine strains of Saccharomyces cerevisiae (flor strains) is a form of cellular aggregation observed as an air-liquid interfacial biofilm at the end of the alcoholic fermentation. Formation of the biofilm appears to be an adaptive mechanism because it ensures access to oxygen and therefore permits continued growth on nonfermentable ethanol. In general, nonbuoyant cells cease growth at the end of completed wine fermentations not for lack of carbon but for lack of oxygen. Biofilm cells have been found to have an elevated and/or altered lipid content and increased surface hydrophobicity (3,5,8,9,11). While both Hsp12, a small heat shock protein (13), and Muc1 (also known as Flo11), a hydrophobic cell wall mannoprotein (4, 6), have been shown to be required for the flor biofilm (10,12,14), other genetic or environmental requirements, other than an absence of glucose and the presence of ethanol and oxygen, have not been demonstrated. Here, we asked whether flor formation could be induced during growth on nonfermentable substrates other than ethanol. On the basis of dry weight of biofilm formed per mg of available carbon, the best carbon sources were found to be glycerol, ethyl acetate, and ethanol, in descending order. While subsurface growth occurred on acetic, DL-lactic, and succinic acids, an air-liquid interfacial biofilm did not always form. Microarray analysis of cells shifted from growth on glucose to growth on ethanol did not detect significant changes in expression of known biofilm formation-associated genes.Yeast strain, growth conditions, and quantitation of biofilm. A flor strain of S. cerevisiae, 3238-32 MATa leu2-⌬1 lys2-801 ura3-52 (12), was grown for 24 h in YEPD (2% Bacto peptone, 1% yeast extract, 2% glucose) at 30°C and 200 rpm. Cells were harvested and washed twice in sterile distilled water by centrifugation, resuspended in sterile distilled water, and diluted 500-fold into sterile-filtered YNB (Bacto yeast nitrogen base without amino acids) plus 30 g/ml Leu, 30 g/ml Lys, and 10 g/ml Ura and supplemented with 4% (vol/vol) ethanol (YNBEtOH), 3% (vol/vol) glycerol (YNB-Glyc), 1% (wt/vol) potassium acetate (YNB-Acet), 2% (wt/vol) DL-lactic acid (YNBLact), 2% (wt/vol) succinic acid (YNB-Succ), or 2% (vol/vol) ethyl acetate (YNB-EtAc). All YNB-based media were adjusted to pH 5.4. Four replicate 1-ml/well cultures were grown in a 24-well polystyrene microtiter plate (Becton Dickinson Labware, NJ). Biofilms were harvested by aspiration after 5 days of static incubation at 30°C by collecting the 1-ml cultures and an additional 1 ml of sterile distilled water used to rinse each well. The 2-ml samples were transferred to a cuvette and read at A 600 . Biofilm bio...

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

“…The remaining biofilm cells were recovered as described above, without touching the bottom of the well, and transferred to the cuvette. The optical density of the final volume in the cuvette (2 ml) was measured spectrophotometrically and the OD 600 value converted to dry weight (mg) (37).…”

“…Biofilm dry weight measurement was carried out essentially as described previously (37), with minor modifications. After 5 days of static incubation at 30°C, biofilm biomass was harvested by pipetting ϳ1 ml of sample per well up and down 3 times (or more times, if necessary) without allowing the pipette tip to touch the bottom of the well, to avoid recovery of non-biofilm-forming cells that had settled at the bottom of each well.…”

The Microbial Diversity of Sherry Wines

“…Flor yeasts are capable of growing on non-fermentable carbon sources, such as glycerol, ethyl acetate, and even ethanol [16]. On the other side, biofilm formation is limited by the presence of high contents of succinic acid, lactic acid, and acetic acid.…”

“…In 1963, Rocques, according to the previous observations of Louis Pasteur, described Mycoderma vini as the yeast species responsible of the veil formed in the yellow wines (vin jaune) from Arbois (Jura region, France). Since then, veil-forming yeast taxonomy and the repercussion of the physiology of the different species over the main components of Sherry wines, have been studied by numerous researchers [1,2,5,6,8,9,15,16]. In the case of flor yeasts, the most commonly analyzed substrates for their differential fermentation and assimilation capabilities are galactose, dextrose, lactose, maltose, melibiose, raffinose, and sucrose [17].…”

Flor Yeast Proteomic Response to the Lack of Fermentable Carbon Source

“…One of the principal is the lack of a fermentable carbon sources like glucose or fructose already consumed during the previous fermentation process. Zara et al, (2010) considered the formation of the biofilm (also called "flor" or "velum") an adaptation system of the flor yeast to a medium which lacks a fermentable carbon source. Biofilm formation in the wine-air area allows the yeast to reach an cold acetone to the supernatant.…”